1

UNIVERSITY OF GENEVA FACULTY OF MEDICINE

DEPARTMENT OF NEUROSCIENCES

KAUNAS UNIVERSITY OF MEDICINE FACULTY OF PHARMACY

DEPARTMENT OF DRUG TECHNOLOGY AND SOCIAL PHARMACY

NEW POTENTIAL PHARMACEUTICAL TARGETS IN EPENDYMAL CELLS:

RESEARCH AND EVALUATION MASTER THESIS

MINDAUGAS JONIKAS FACULTY OF PHARMACY

KAUNAS UNIVERSITY OF MEDICINE

SUPERVISORS:

PROF. JOZSEF ZOLTAN KISS PROF. VITALIS BRIEDIS DEPARTMENT OF NEUROSCIENCES, DEPARTMENT OF DRUG TECHNOLOGY

FACULTY OF MEDICINE, AND SOCIAL PHARMACY,

UNIVERSITY OF GENEVA. FACULTY OF PHARMACY,

KAUNAS UNIVERSITY OF MEDICINE.

GENEVA, KAUNAS

2010

2

Table of Contents

1. The goal of study……..……………………………………………….14

2. Introduction…………………………………..…..…………………..15

2.1 Ependymal cells…………………………………………….………….……15

2.1.1 Cilia ………………………………………………………………………......…..16

2.1.2 Microvilli…………………………………………………………………...…….20

2.1.3 Adhesion…………………………………………………………...…………….20

2.1.4 Junctions…………………………………………………………………..……..21

2.1.5 Development…………………………………………………….…….…………22

2.1.6 The support of the neurogenic niche………………………….……….…………23

2.1.7 Other cells………………………………………………………..….……………25

2.2 Wnt Signaling system…………………………………………….………...26

2.2.1 Wnt biogenesis……………………………………………….……….………….28

2.2.2 Receptors in Wnt signaling………………………………………………………29

2.2.3 Wnt signaling and adult neurogenesis……………………………..……………..31

2.2.4 Wnt signalling and Alzheimer disease (AD)…………………………………….32

2.2.4 The structure of Dvl………………………………………….…………………..35

2.2.5 Dishevelled controls apical docking of basal bodies and planar cell

polarization………………………………………………………………….…….…... 37

3. Experimental part……………………………………………………40

3.1 Materials and methods……………………………………………………. 40

3.1.1 Cell culture………………………………………………………………………. 40

3.1.2 Brain slices……………………………………………….……………………….42

3

3.1.3 Analysis……………………………………….………………………………..45

3.2 Results……………………………………………………………………. 46

3.2.1 Analysis of Wnt ligands………………………………………………………...46

3.2.2 Analysis of Wnt signal transducer – Dishevelled……………………………….49

3.3 Discussion…………………………………………………………………..53

3.3.1 Ependymal cells can secrete Wnt 8b.....................................................................54

3.3.2 The role of Dishevelled as Wnt signal transducer in the cilia………………….. 56

3.3.3 Dishevelled nuclear shuttling…………………………………………………... 57

4. Conclusions..........................................................................................59

5. Bibliography:…………………………………….…………..............62

4

SANTRAUKA

NAUJI POTENCIAL�S VAIST� TAIKINIAI

EPENDIMIN�SE L�STEL�SE: TYRIMAI IR VERTINIMAS

Darbo tikslas:

Atlikti tyrimus, siekiant surasti galimus naujus vaist� taikinius ependimin�se l�stel�se.

Tyrimo objektas:

1. Suagusi� žiurki� smegen� skilvelin�s zonos l�stel�s bei ependemini� l�steli�

kult�ra.

2. Wnt signalinio kelio molekul�s.

Tyrimo metodai:

1. Specifiniai fluorescenciniai imunohistocheminiai tyrimai atliekami su smegen�

skilvelin�s zonos l�stel�mis ir ependemini� l�steli� kult�ra.

2. Rezultat� analizei naudojamas konfokalinis ir fluorescencinis mikroskopai.

Darbo uždaviniai:

1. Suformuoti model� tinkam� tirti suaugusi� žiurki� smegen� skilvelin�s zonos

l�steles.

2. Sukurti ependimini� l�steli� kult�ros model�.

3. Nustatyti Wnt signalinio kelio ligand� (Wnt 3A, Wnt 5A, Wnt 7A, Wnt 7B ir Wnt

8b) lokalizacij� suaugusi� žiurki� smegen� skilvelin�s zonos l�stel�se ir

ependimini� l�steli� kult�rose. Tam panaudoti specifinius imunohistocheminius

metodus.

4. Siekiant išsamesni� rezultat�, taip pat lokalizuoti Dishevelled baltymo izoformas

(Dvl-1, Dvl-2 ir Dvl-3) suaugusi� žiurki� smegen� skilvelin�s zonos l�stel�se ir

ependimini� l�steli� kult�rose. Tam pasiekti, bus pasitelkti specifiniai

imunohistocheminiai metodai.

5

Rezultatai:

1. Wnt 3A, Wnt 5A, Wnt 7A ir Wnt 7B antik�ni� specifin�s imunin�s reakcijos

ependimin�se l�stel�se neaptikome taikant konfokalin� ir fluorescencin�

mikroskopus.

2. Wnt 8b antik�nio specifin� imunin� reakcija aptikta apatin�je blakstien�li� dalyje.

3. Dvl-1 epedimin�se l�stel�se yra akumuliuojamas apatin�je blakstien�l�s dalyje ir

l�stel�s branduolyje. Tai nustat�me pagal specifin� imunin� antik�nio reakcij�.

4. Dvl-2 antik�nis nepasižym�jo specifine imunine reakcija ependimin�se l�stel�se.

5. Dvl-3 ependimin�se l�stel�se nustat�me membranoje ir nedidelis kiekis galimas

citoplazmoje.

1. Lentel�. Specifin� lokalizacija Wnt signalin�s sistemos ligand� ir signalo pardav�j�.

Wnt3A

Wnt5A

Wnt7A

Wnt7B

Wnt8B

Dvl-1

Dvl-2

Dvl-3

Ependemini� l�steli� kult�ra: • Blakstien�les

• Citoplazma

• Branduolys

• Membrana

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

+++

-

-

-

++

+

+++

-

+/-

+/-

+/-

+/-

-

++

+/-

++

Skilvelin�s zonos l�stel�s: • Blakstien�les

• Citoplazma

• Branduolys

• Membrana

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

+++

-

-

-

++

+/-

+++

-

-

-

-

-

-

++

-

+++

- - n�ra signalo; +/- - labai silpnas ar nespecifiškas signalas; + - silpnas signalas; ++ - geras signalas; +++ - stiprus signalas.

6

Išvados:

1. Suaugusi� žiurki� smegen� skilvelin�s zonos l�steles galima tirti smegen�

segmentuose, kurie yra gaunami „Vibratomo“ ar „Kryostato“ pagalba. Taikant šiuos

metodus, l�steli� pozicija yra išsaugojama ir strukt�ra nepažeidžiama; tokios

l�stel�s yra tinkamos imunohistocheminiams tyrimams. Optimaliausias segment�

storis yra 60-20µm.

2. Ependimini� l�steli� kult�ra gali b�ti sukuriama iš k� tik gimusi� žiurki� jaunikli�

smegen�. Ependimin�s l�stel�s yra išpjaunamos iš galvos smegen� tre�iojo

skilvelio ir kultivuojamos Lamininu padengtose l�kštel�se. Kultivavimas vykdomas

7-10 dien�. Atlikus l�steli� fiksacij�, galimi imunohistocheminiai tyrimai.

3. Wnt 3A, Wnt 5A, Wnt 7A ir Wnt 7B neaptikome ependimin�se l�stel�se. Pagal

šiuos rezultatus galime teigti, kad šie ligandai negali b�ti vaist� taikiniai, nes

tikriausiai n�ra ekspresuojami ependimin�se l�stel�se. Yra tikslinga šiuos

duomenis patvirtinti in situ hibridizacijos metodu.

4. Wnt 8b yra susikaup�s apatin�je blakstien�li� dalyje, ši lokalizacija yra visiškai

nauja. Ši blakstien�li� dalis pasižymi sekrecin�mis funkcijomis, tod�l manome, kad

Wnt 8b gali b�ti sekretuojamas � galvos smegen� skyst�. Tai gali b�ti labai svarbu

prenataliniam smegen� žiev�s vystimuisi ir regeneracijai po smegen� pažeidimo.

Wnt 8b galimas labai svarbus naujas vaist� taikinys.

5. Dvl -1 lokalizuota ependimini� l�steli� apatin�je blakstien�li� dalyje ir branduolyje.

Ši informacija literat�roje dar nemin�ta. Pagal Dvl-1 viet�, galima spr�sti, kad jis

dalyvauja tipiniame Wnt signaliniame kelyje. Dvl-1 gali b�ti svarbus vaist� taikinys

gydant ependimomas, nes yra žinoma, kad kitose v�žio formose tipinis Wnt

signalinis kelias yra pernelyg suaktyvintas. Ependimomose Dvl-1 gali b�ti

inhibuojamas vaistais, siekiant subalansuoti tipin� Wnt signalin� keli�.

6. Dvl-2 neaptikome ependimin�se l�stel�se, taip pat literat�roje n�ra duomen�, kad

šis baltymas yra ekspresuojamas šiose l�stel�se. Pagal šiuos duomenis galime teigti,

kad Dvl-2 negali b�ti vaist� taikinys. Yra tikslinga šiuos duomenis patvirtinti in situ

hibridizacijos metodu.

7

7. Ependimin�se l�stel�se Dvl-3 yra akumuliuojamas membranoje. Ši akumuliacijos

vieta yra visiškai nauja. Dvl-3 lokalizacija membranoje rodo jo dalyvavim� ne-

tipiniame Wnt signaliniame kelyje, kuris paveikia l�stel�s poliarizacija. Pagal šiuos

duomenis galime teigti, kad Dvl-3 gali b�ti geras vaist� taikinys gydant ligas,

kurioms pasireiškia sutrikusi blakstien�li� veikla. Kaip pavyzdys gal�t� b�ti,

sutrikusi neuron� migracija lygiagre�iai šoninio skilvelio. Tod�l, Dvl-3 gali b�ti

geras taikinys koreguojantis degeneracinius procesus, nes didesn� ekspresija Dvl-3

pagreitint� neuroblast� ir astrocit� migracij� link pažeistos vietos, taip b�t�

sustabdomi tolimesni pažeidimai.

8

SUMMARY

NEW POTENTIAL PHARMACEUTICAL TARGETS IN

EPENDYMAL CELLS: RESEARCH AND EVALUATION

The aim:

Conduct an investigation study in order to find potential new drugs targets in ependymal

cells.

Objects:

1. Ventricular zone cells of adult rat animal and ependymal cell culture.

2. WNT signaling pathway molecules.

Methods:

1. Specific fluorescent immunohistochemistry studies performed with brain

ventricular zone cells and ependymal cell culture.

2. Analysis of the results was done with confocal and fluorescence microscopes.

The objectives:

1. Develop an effective model to investigate the ventricular zone cells of adult rat

brain.

2. Establish the model of ependymal cell culture.

3. To determine the localization of the WNT signaling pathway ligands (WNT 3A,

WNT 5A, WNT 7B, WNT 7A, and WNT 8b) in ventricular zone cells of adult rat

brain and in ependymal cell culture. In order to accomplish this, use specific

immunohistochemical methods.

4. With a purpose to achieve more comprehensive result, localize Dishevelled protein

isoforms (Dvl-1, Dvl-2 and Dvl-3) in ventricular zone cells of adult rat brain and in

ependymal cell culture. In order to do this, use specific immunohistochemical

methods.

9

Results:

1. Appling confocal and fluorescence microscope specific immune response was not

observed of WNT 3A, WNT 5A, WNT 7A and WNT 7B antibodies in ependymal

cells.

2. Specific imunoreactivity of Wnt 8b was detected in the basal part of the cilia.

3. Dvl-1 is accumulated in the basal part of cilia and in nucleus of the ependymal

cells.

4. Dvl-2 antibody did not exhibit specific immune response in the ependymal cells.

5. Dvl-3 was detected on the membrane and low amount in the cytoplasm of

ependymal cells.

Table 1. Specific localization of Wnt signaling pathway ligands and signal transmitters.

Wnt3A

Wnt5A

Wnt7A

Wnt7B

Wnt8B

Dvl-1

Dvl-2

Dvl-3

Cultured rat ependymal cells: • Cilia

• Cytoplasm

• Nucleus

• Membrane

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

+++

-

-

-

++

+

+++

-

+/-

+/-

+/-

+/-

-

++

+/-

++

Rat brain slices: • Cilia

• Cytoplasm

• Nucleus

• Membrane

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

+++

-

-

-

++

+/-

+++

-

-

-

-

-

-

++

-

+++

- - no signal;

+/- - very weak or uspecific signal;

+ - weak signal;

++ - good signal;

+++ - Strong signal.

56

Conclusion:

1. Ventricular zone brain cells of adult rat brain can be studied in a sections,

obtained by "Vibratome" and "Cryostat”. Using these techniques, the position

and structure of the cells is maintained; these cells are suitable for

immunohistochemical experiments. Optimal thickness of sections is 60-20�m.

2. The culture of ependymal cells can be created from new-born baby rat brain.

Ependymal cells are dissected out of the third ventricle of the brain and cultured

in Laminin coated dishes. Cultivation is carried out for 7-10 days.

Immunohistochemical experiments can be done after cell fixation.

3. We did not detect WNT 3A, WNT 5A, WNT 7A and WNT 7B in the ependymal

cells. According to these results, we suggest that these ligands can not be targets

for the drugs, because it might be that there is no expression of these proteins in

ependymal cells. It is advisable to confirm these results with situ hybridization

method.

4. WNT 8b is accumulated in the basal part of the cilia, this is brand new location.

It is known, that this part of the cilia possesses secretion features that is why we

believe that WNT 8b may be secreted from ependymal cells to the cerebro spinal

fluid. This may be very important to prenatal cortical development and

regeneration after brain damage. WNT 8b may be an important new drug target.

5. Dvl-1 localization in the nucleus and basal part of cilia of ependymal cells is not

reported in the literature. According to Dvl-1 accumulation place, we can

suggest that it participates in canonical WNT signaling. Dvl-1 may be an

important drug target for treating ependymomas, because in other forms of

cancer, it is known that canonical WNT signaling pathway is over activated. In

ependymomas Dvl-1 may be drug inhibited in order to restore balance of

canonical Wnt signaling.

6. Dvl-2 was not detected in the ependymal cells, as well there is no evidence in

literature that this protein is expressed these cells. According to this data, we

strongly suggest that Dvl-2 can act as a drug target. It is advisable to confirm

these results with situ hybridization method.

7. Dvl-3 is accumulated on the membrane of the ependymal cells. This localization

is a completely new. Dvl-3 membrane localization suggests its involvement in

11

non-canonical WNT signaling, which affects planar cell polarization. According

to these data, Dvl-3 may be a good drug target for treating the diseases, which

display the impaired cell migration or ciliar activity. An example could be

impaired neuronal migration in parallel with the lateral ventricle. Thus, Dvl-3

may be a good target for adjusting the degenerative processes, because increased

expression of Dvl-3 might accelerate migration of neuroblasts and astrocyte

toward damaged areas, thereby stopping further damage.

12

LIST OF ABBREVATION:

• AChR - acetylcholine receptor.

• AD - Alzheimer disease.

• AMPA - -amino-3-hydroxyl-5-methyl-4-isoxazole-propionate.

• Ang-1 – angiopoietin.

• APC - adenomatous polyposis coli

• APP - amyloid precursor protein.

• AQP – Aquaporins.

• BBS4 - Bardet-Biedl syndrome 4 protein.

• BSA – bovine serum albumin

• CamK2 - calcium-calmodulin-dependent kinase 2

• CSF - cerebro spinal fluid

• CVO - circumventricular organs • CRD - cysteine-rich domain

• COMT - catechol O-methyltransferase.

• Dvl – Dishevelled • Evi - Evenness interrupted

• FGF - Fibroblast growth factors.

• Fz – Frizzleds

• GEF - guanine nucleotide exchange factor • GLUT - Glucose transporters.

• GSK3 - glycogen synthase kinase 3

• GTPase - family of hydrolase enzymes,which bind and hydrolyze guanosine

triphosphate.

• IGF - insulin-like growth factor

• IDA - inner dynein arms

• JNK - Jun kinase

• LEF/TCF - lymphoid enhancer factor/T cell factor

13

• LRP - lipoprotein receptor related protein

• MIF - migration inhibitory factor

• NCAM - neural cell adhesion molecule.

• NES - nuclear export sequence

• NET - norepinephrine transporters.

• NEuROD1 - pro-neurogenic transcription factor

• Nkd - Naked Cuticle.

• NMDA - N-methyl-D-aspartic acid.

• NSC - Neural stem cells.

• ODA - outer dynein arms • PAF – paraformaldehyde

• PBS - Phosphate buffered saline

• PCP – planar cell polarity.

• PKC - protein kinase C

• RPGR - retinitis pigmentosa GTPase regulator

• sFRP - Secreted frizzled-related protein

• SGZ - subgranular zone

• Srt – Sprinter

• SVZ - Subventricular zone.

• TRPV4 - Transient receptor potential cation channel subfamily V member 4.

• T2R - Taste Receptor 2.

• VEGF - Vascular endothelial growth factor.

• WG – Wingless

8.

14

1. The goal of study

The goal of my study is to investigate Wnt signaling in the ventricular zone and to

localize Wnt signaling elements (ligands and transducers) using immonohistochemistry.

WNT ligands are secreted proteins that regulate cell fate decisions, cell polarity, cell

migration, axonal morphology, and synaptic differentiation. In mammals, complexity

and specificity of Wnt signaling are in part achieved through 19 Wnt ligands. Several

studies already reported that Wnt genes continue to be expressed in the adult brain.

However, there is no immunoreactivy studies done concerning ependymal cells and Wnt

signaling elements. Therefore, I investigated the localization of Wnt elements

immunoreactivity with respect to the cilia and other structures of ependymal cells, in

order to find new drugs targets.

1. I had chosen to explore these ligands: Wnt 3A, Wnt 5A, Wnt 7A, Wnt 7B

and Wnt 8b.

2. In order to achieve comprehensive results, I had also investigated wnt

signal transmitter – Dishevelled.

As investigation object I used in vitro model of subventricular zone-derived

ependymal cells and brain section in vivo.

15

2. Introduction 2.1 Ependymal cells The ependyma is an

uninterrupted, single-cell

epithelium layer, composed of

neuroglial cells, mainly ciliated

ependymal cells. The character of

the epithelial lining was first

documented by Purkinje (1836),

and investigators subsequently

recognized that the ependyma is

heterogeneously composed, in

particular that some cells had basal

processes that extended into the

subjacent neuropil (Agduhr,

1932; Wislocki, 1932). In 1954,

Horstmann first applied the

descriptive term tanycyte to such

elongated ependymal cells. The

ependyma layer covers the entire

ventricular system of brains and

the central canal of the spinal

cord. The ependymal layer in the

adult is relatively uniform, but there are some specialized places: the area postrema at

the caudal end of the floor of the fourth ventricle and subcommissural organ at the

transitional zone between the roof of the third ventricle and the cerebral aqueduct. Those

places distinguish by a lack of the cilia and of blood-brain barrier. The ependyma at the

floor of the third ventricle becomes modified in early fetal life and also lacks blood-

brain barrier.

Figure 1. Features of normal ependymal. A. Transmission electron micrograph showing mature ependymal cells of the lateral ventricle. The top right inset shows a supraependymal axon. Bar 0.25 µm. B. Scanning electron micrograph of the surface of the caudate nucleus of an adult human showing the dense packing of cilia clusters. (Bigio et al., 2009)

16

The ependyma principally consist of ependymal cells, which develop from

neuroectoderm. Ependymal cells are neuroglial cells, morphology can be described as

cuboidal to columnar shape and a fairly round nucleus with fine stippled chromatin

pattern and inconspicuous nucleolus. The surface is covered by microvilli and most of

the cells have a central cluster of long motile cilia.

Mature mammalian ependymal cells possess the structural and enzymatic

characteristics necessary for scavenging and detoxifying a wide variety of substances in

the cerebro spinal fluid (CSF) and it is forming a metabolic barrier at the brain–CSF

interface. The presence of motile cilia, microvilli, and zonula adherens junctions at the

apical surfaces is important for these roles. The gap junctions might be used to

coordinating the cells activity.

2.1.1 Cilia

History. The field of ciliary biology is an active area of study with a rich history.

It might be, that cilia were observed first time and their motile function assessed by

Antoni Va Leeuwenhoek in 1674-75 (Dobell, 1932), but these organelles were named

by Otto Friedrich Müller in 1786 (Muller, 1786). In the second half of the nineteenth

century the non-motile cilia were observed (Kowalevsky, 1867; Langerhans, 1876;

Zimmermann, 1898). Zimmermann was the first scientist to observe these organelles in

mammalian cells, including those of humans. He named those organelles – central

flagella (“centralgeissel”) and hypothesized that they have a sensory function. However,

both Zimmermann’s name for these organelles and his proposed function for them were

soon forgotten. Sorokin in 1968 renamed these organelles “primary cilia” (Sorokin,

1968). This special class of non-motile cilia, because of their evident sensory functions,

was investigated deeper than motile cilia. Currently, however, a new idea in the field of

cilia is emerging – that all cilia have sensory functions (Christensen et al., 2007).

Evidence supporting a sensory role for motile cilia has been accumulating in the

literature for a very long time.

Structure. A striking feature of the ependymal cells is the apical cluster of the

motile cilia, which projects into the CSF. Cilia are microtubule-based cell organelles

17

extending from a basal body, a

centriole, at the apical cell

surface, containing 9+2

axonemes surrounded by a

specialized ciliary membrane.

This microtubule scaffold,

(also known axoneme), is

enveloped by an extension of

the plasma membrane. The

structure is retained in place by

a number of other associated

proteins, like the radial spoke

proteins, which help to

connect the peripheral

doublet microtubules with the

central pair. Major structures that attach to the microtubules, the outer and inner dynein

arms (ODAs and IDAs), the radial spokes, the central-pair projections, and so forth are

defined protein complexes. The ODAs and IDAs are force-producing molecular motors

that cause the doublet microtubules to slide with respect to one another. The doublet

sliding is asynchronous with the progression of activity around the axoneme, yielding a

helical beat. Maximum beat frequencies range up to approximately 100 Hz, although

most reports of mammalian ciliary beat frequency are much lower, perhaps normally

10–20 Hz.

Below plasma membrane, the axoneme remains anchored to the basal body, which

is derived from the mother centriole. The basal body is a template on which the

axoneme is built by intraflagellar transport, an intracellular cargo delivery system. The

building starts from bringing preassembled axonemal components from cytoplasm to the

tip of the axoneme. Ependymal cells have an additional kind of cilia called primary cilia.

These cilia are relatively short in size and lack the central pair of singlet microtubules as

well as dynein arms (hence they are immotile). Primary cilia appear to be associated

almost exclusively with sensory functions.

Figure 2. Basic ciliary structure. Schematic representation of a cilium and cross-section of a basal body composed of microtubule triplets and a ‘‘9+2’’ and a ‘‘9+0’’ axoneme showing the position of dynein arms and radial spokes needed for force generation and coordination. Along the outer microtubule doublets of the axoneme, molecular motors transport IFT particles. (Rodriguez et al., 2009)

18

Dynamic analysis of living ependyma shows that the cilia are beating in a

coordinated manner, trending to sweep foreign particles in the same direction as bulk

CSF flow (Yamadori and Nara, 1979). The coordination of cilia beating might be

accomplishment by communication through gap junctions or through innervations. The

CSF is also stirred at the ependymal cells surface to facilitate metabolic interactions

between ependyma and CSF content (Roth et al., 1985).

The cillary necklace. Specific proteins are localized to or concentrated in the

ciliary membrane, as opposed to the rest of the cell membrane. There are some

hypotheses that there is a selective barrier at the cilium entrance. Mode of operation of

the barrier is still uncertain. This specialized barrier region is found on all 9+2 and 9+0

mammalian and invertebrate cilia that have been studied by freeze fracture electron

microscopy (Gilula and Satir, 1972). Transport proteins have been localized to the

necklace region near the repeating intersection of particles and the membrane (Gilula

and Satir, 1972), which may imply that the particle rich regions are assembly sites for

transport of membrane and axonemal cargos.

The membrane in the necklace region of airway cilia has a different composition in

terms of lectin binding, anionic charge and free-cholesterol distribution compared to the

rest of the ciliary and cell membrane (Tuomanen, 1990). A putative guanine nucleotide

exchange factor (GEF), retinitis pigmentosa GTPase regulator (RPGR) and its

interacting protein are localized to a necklace of the connecting cilium of the

photoreceptor (Hong, 2001). RPGR isoforms are also found in the necklace region of

motile cilia of the trachea (Hong, 2003).

The importance of the necklace in tracheal cilia is reemphasizing by its disruption

and disappearance upon infection after attachment of these bacteria to the cilium and

prior to cell death. Moreover, when cilia are shed, the point of breakage and membrane

resealing occurs just above the ciliary necklace and the necklace persists.

Membrane. 9+2 and 9+0 cilia axonemes are surrounded by a ciliary membrane,

which extends from the cell membrane but is selectively different from the cell

membrane in overall composition. Surprisingly, was known very little about the ciliary

membrane until quite recently. Now, through cilia fractionation and proteomics, the

composition of the membrane proteins of the cilium is emerging.

19

Cilia response pathways for unicellular organisms are necessary for survival,

motion, control of the 9+2 motile cilium. The activity of this pathway depends on

specific receptors and channel proteins, like: cyclic nucleotide receptor, Ca2 + channels

and receptors involved in growth control pathways. All those above motioned structures

are localized to the ciliary membrane (Satir and Guerra, 2003). This suggests that all

cilia have sensory function and this hypothesis will be supported below, by discussing

studies in mammals.

Sensory reception. Motile cilia of the mammalian respiratory epithelium have

been reported to show mechanosensitivity and chemosensitivity. As mucus viscosity

around the cilia increases, ciliary mechanics are adjusted so as to maintain a ciliary beat

frequency, although it is reduced, but still sufficient to maintain transport of mucus to

the larynx, where it will be swallowed or expectorated (Johnson et al., 1991). Cytosolic

Ca2+ levels play an important role in this process of autoregulation. Increased Ca2+

concentration in cytoplasm are associated with changes in ciliary beat frequency

(Salathe, 2006).

Sanderson and Dirksen observed that mechanical stimulation of cilia, of cultured

rabbit tracheal cells with fluid movement, induced a transient increase in ciliary beat

frequency, it was dependent on the presence of Ca2+ in the extracellular medium and was

inhibited by a Ca2+-channel blocker (Sanderson and Dirksen, 1986). Lorenzo and

colleagues localized the TRPV4 cation channel in the cilia of respiratory epithelial cells

and showed that it was lost from the respiratory cilia of TRPV4 knockout mice (Lorenzo

et al., 2008). A TRPV4 agonist induced Ca2+ influx and an increase in ciliary beat

frequency in the tracheal epithelial cells from Trpv4+/+ mice but not Trpv4–/– mice. I

have to mention, that wild-type and Trpv4–/– cells were able to autoregulate ciliary beat

frequency in response to a viscous load, but just wild type cells were able to show

normal intracellular Ca2+ oscillations.

Shah and colleagues recently reported a chemoreception when they localized

different members of the bitter taste receptor family to motile cilia of airway epithelial

cells (Shah et al., 2009). There are bitter compounds, which are known to be acting

through T2R receptor and activate a signaling pathway, which induces the G protein -

gustducin (localized to the cilia), phospholipase C2 and a rise in cytosolic Ca2+. Shah

and colleagues applied those compounds to the ciliated cells. The results were induced a

20

transient, dose-dependent rise in intracellular Ca2+ in ciliated cells as well as an increase

in ciliary beat frequency.

There is evidence that than pathogens bind to respiratory cilia, the signaling

pathways can be activated: binding of Mycoplasma hyopneumoniae to the ciliary

membrane was shown to activate a G-protein-coupled receptor, which activates a

phospholipase C pathway that results in a rise in intracellular Ca2+ (Park et al., 2002).

Polycystins are now commonly known to be present in ciliary membranes of 9+0

primary cilia (Geng, 2006).It might be that similar types of channels and receptors are

present in the membranes of all mammalian motile cilia and that they play a role in

epithelial homeostasis.

The resemblance of sensory function among nearly all eukaryotic cilia (both

primary cilia and motile cilia) suggests that the original protocilium from which both

types of organelles evolved was a sensory organelle. Cilia might have evolved from a

simple „sensory membrane patch“(Jekely and Arendt, 2006; Satir et al., 2008) which

during evolutionary time extended to antenna-like structure, arising from the cell surface

and only later acquired motile function. Obviously, both sensory reception and motility

provided selective advantages for the early eukaryotic cell.

2.1.2 Microvilli

The apical surface of ependymal cells is covered by microvilli. Microvilli are a

microstructure covered with a plasma membrane, which encloses cytoplasm and

microfilaments. Although microvilli are cellular extensions, there are no or little cellular

organelles in them. Microvilli of ependymal cells are covered with glycocalyx coating.

The studies revealed presence of sialic acid, poly-N-lactosamine and D-galactose on the

ependymal microvilli (Acarin et al. 1994; Adam et al. 1993). Those enzymes along

glycoproteins are important components of chemical recognition and information

transfer mechanisms on the cell surface (Paulson, 1989). Thus the surface of ependymal

cells is adapted to interact chemically with CSF.

2.1.3 Adhesion

21

Cells in the ependyma layer are bounded to their neighbour at the apical surface by

zonula adherens type junctions (Brightman and Reese, 1969). There are tight junctions

only in between of ependymal cells covering specialized circumventricular organs

(CVO), including the choroid plexus, because there capillaries lack tight junctions.

Adherens junctions consist of cadherins, calcium-dependent transmembrane

adhesion molecules. The cadherin substances family includes cadherins,

protocadherins, desmogleins and desmocollins. On the extracellular surface cadherins

bind each other homotypically, while the intracellular domains bind p120-catenin and -

catenin (Redies et al. 1996). Numb and Numbl are required for maintenance of cadherin

based adhesion and polarity of radial glia and ependyma. In addition to apical adhesive

molecules human and mice ependymal cells reported to be expressing neural cell

adhesion molecule (NCAM, also called CD56), homophilic binding glycoprotein

(Figarella-Branger D. 1995)

Ependymal cells have a basal lamina between them (Bruni JE. 1998). The basal

lamina is a layer of extracellular matrix approximately, 40 -50 nanometers, on which

ependymal cells sticks. This layer is secreted by the ependymal cells. The basal lamina

consists of a complex of substances: laminin, utrophin, alpha-dystrobrevin and beta-

dystroglycan. This layer is anchoring ependymal cells to the ventricle wall.

2.1.4 Junctions

The membrane of ependymal cells exhibit several important classes of molecular

channels: gap junctions and aquaporins. Gap junctions forming proteins contribute to:

ion homeostasis, volume control, transferring electric current, intracellular, mechanical

sense and supporting adherent connections between neighbouring cells. Occasionally

gap junctions can be formed between ependymal cells and adjacent astrocytes. Gap

junctions are formed by hemichannels (connexons), which consist of an oligomer of six

proteins (connenxins). A complete gap junction channel is formed by two hemichannels

in mirror symmetry. (For review see Prochnow and Dermietzel, 2008). The studies of

gap junction’s proteins revealed conexins 26 and 43 in the ependymal cells. There is

data showing that ependymal cells express connexin 32 (for review see Bigio, 1995).

Colocalization of connexin 43 and basic fibroblast growth factor has been postulated to

22

play an important role in the regulation in the regulation of gap junction

communications (Yamamoto et al., 1991).

Aquaporins (AQP) are molecular channels. They are formed by two tandem

repeats of three membrane-spanning -helices, with cytoplasmic carboxyl- and

aminoterminals. Two connecting loops, each containing an Asn-Pro-Ala motif, are

believed to determine water selectivity (Murata et al., 2000). The expression and

distribution of aquaporins are organized to control water movements in the brain

(Badaut, 2002). Aquaporin 4 is expressed in the ependymal cells at the basolateral

aspects of ependymal cells, as well as in the end feet of most astrocytes (Li, 2009).

AQP4 might have structural functions beyond its role as a water channel, because AQP4

knockout mice develop hydrocephalus secondary decrease in the expression of Cx43

and loss of lateral junctions leading to disruption of the ependymal lining (for review see

Bigio, 1995). AQP1 and AQP2 immunoreactivities have been documented in human

ependymal cells (Mobasheri et al., 2004, Mobasheri et al. 2005).

2.1.5 Development

During early neurodevelopment, the embryonic ventricles are lined by a

germinal epithelium. This embryonic neuroepithelium has planar polarity that drives

morphogenetic movements essential for neural tube closure (Colas and Schoenwolf,

2001; Wallingford, 2006). Radial glial cells, in this epithelium, contain both spatial and

temporal patterning that determines cell fate and cell position in the developing brain

(Hebert and Fishell, 2008). A subpopulation of radial glia transform into ependymal

cells (Spassky et al., 2005). The majority of the ependymal cells undergo their final

nuclear DNA synthesis between E14 and E16 (Spassky et al, 2005). The ciliogenesis of

ependymal cells begins around birth and progresses in a direction from caudal to rostral

and from ventral to dorsal along the lateral wall of the lateral ventricles of the rodent

brain (Sarnat, 1992; Bruni, 1998). Spassky with others showed that ependymal cilia

appeared between P0 and P4 (Spassky et al, 2005).

The ependymal do not divide in the adult. The postmitotic characteristic of

mature ependymal cells is confirmed by studies showing that multiciliated epithelial

cells with numerous basal bodies in the apical cytoplasm are postmitotic (Lange et al.,

23

2000) and the ependymal layer in the mammalian brain does not regenerate when it is

injured (Sarnat, 1995).

2.1.6 The support of the neurogenic niche

The close contact between

ciliated ependymal cells and

pluripotent cells has led to

investigation of ependymal cells as a

modulator of stem cell populations.

The ependymal cells maintain basal

processes in regions of the frontal

horns where SVZ cells are most

abundant (Rodriguez et al., 2003).

Recently, Mirzadeh with other

observed that ependymal cells form a

remarkable pinwheel organization

specific to regions of adult

neurogenesis. The pinwheel’s center

contains the apical endings of the

neural stem cells (NSCs, B1 cells) with

a direct contact to the ventricle and a

long basal process ending on blood

vessels. The peripheral part of the pinwheel consists of two types of ependymal cells:

multiciliated (E1) and a type (E2) characterized by only two cilia and extraordinarily

complex basal bodies (Mirzadeh et al. 2008). These results suggest that adult NSCs

retain fundamental epithelial properties, including apical and basal subdivision.

There are some hypotheses that ependymal cells maintain the SVZ through

production of specific extracellular matrix and adhesion molecules (Hauwel et al., 2005)

or through release of other modulators (Sarnat et al., 1992). Ependymal cells are

producing growth factors. This might contribute to the trophic function of ependymal

cells. Fibroblast growth factors (FGF) are one the most important growth factors in brain

development (Iwata et al., 2009). It has been several times described that mature

Figure 3. Three-dimensional model of the adult SVZ

neurogenic niche illustrating B1 cells (blue; stem cells),

C cells (green; supporting cells), and A cells (red). B1

cells have a long basal process that terminates on blood

vessels (orange) and an apical ending at the ventricle

surface. Note the pinwheel organization composed of

ependymal cells (light and dark brown) encircling B1

apical surfaces. (Mirzadeh et al., 2008).

24

ependymal cells of rodents possess of FGF2 (also known as FGF) (Cuevas et al., 2000;

Fuxe et al., 1996). Some studies reported presence of mRNA for FGF2 in ependyma

(Frautschy et al., 1991), while others observed that most ependymal cells synthesize

FGFreceptor1, which allows resorption and concentration of FGF2 (Gonzalez et al.,

1995). Ependymal cells are also reported to produce FGF1 (acidic FGF) in normal and

pathological conditions (Li et al., 1998; Oomura et al., 1992; Ye et al., 2002).

Hayamizu with colleagues observed an increment of FGF2 following ischemia, this

suggest it has a role in trophic support of adjacent cells (Hayamizu et al., 2001). The

calcium binding protein S100B, which has gliotrophic and neurotrophic properties, is

well known to be present in ependymal cells, particularly at certain times in

development (Sarnat, 1992; Sarnat, 1998; Steiner et al., 2007; Vives et al., 2003)

Vascular endothelial growth factor (VEGF) is expressed by human ependyma

between 22- and 40-weeks gestations (Arai et al., 1998). There are some suggestions

that it has autocrine and paracrine functions. VEGF appears to help for the ependymal

cells to maintain their stuture, because inhibition of VEGF in mice leads to

disappearance of microvilli (Maharaj et al. 2008). VEGF is upregulated in the rat

ependyma following ischemia (Wang et al., 2008). The proteins: Ang-1, Ang-1, Tie-2,

and Flt-1, which are typically associated with endothelial growth, are present in rat

ependymal cells. Several studies have proposed that these proteins might have an

autocrine effect (Horton et al., 2009; Nourhaghighi et al., 2003; Tonchev et al., 2007).

The calcium binding protein S100B, which funtions as gliotrophic and neurotrophic

substance, is already known to be in ependymal cells, especially at particular time points

in development (Sarnat et al., 1992; Steiner et al., 2007). A variety of other growth

factors have been demonstrated in ependymal cells, but the precise functions remain to

be determined.

There are emerging evidences suggesting that ependymal cells have an impact to

the adjacent SVZ cells populations’ through metabolic regulation. Glucose uptake by

ependymal cells from CSF can occur via glucose carriers. The ependymal cells are

reported to possess GLUT1, GLUT2, GLUT3, and GLUT4 (Kobayashi et al.,1996;

Silva-Alvarez et al., 2005; Yu et al 1995). Glucokinase which is responsive to insulin

and insulin-like growth factor (IGF-1) is present in ependymal cells. Normally,

glucokinase acts like a glucose sensor, in other cells type. Glucose might be converted to

25

glycogen, later it can be mobilized by noradrenalin and serotonin, and this means that

ependymal cells might maintain glycogen as a regulated energy store (Prothmann et al.,

2001; Verleysdonk et al., 2005).

2.1.7 Other cells

As described above, ependymal cells are very close to SVZ cells and astrocytes,

but there are several additional populations worth to be mentioned. There are two

neuron systems associated with the ependymal layer. Directly applied to the ventricular

surface of ependymal cells is the supraependymal plexus of serotonergic axons (Brusco,

1998), which arise from the raphe nucleus. Immunohistochemical studies revealed

axons, positive for tyrosine hydroxylase and they are running along basal surface of

ciliated ependyma of lateral ventricles. Presumably these axons contain dopamine and

norepinephrine (Michaloudi, 1996). There are known that ependymal cells express D1

and D2 receptor subtypes, norepinephrine transporters (NETs). Monaamine oxidase B

(MAOB) and catechol O-methyltransferase (COMT) were detected in the ependymal

cells (for review see Bigio, 1995). These innervations together might be helping to

coordinate beating of cilia (Nguyen et al. 2001) or regulate metabolism (Verleysdonk et

al., 2005). In addition, there are widespread system of CSF-contacting neurons that have

extended dendritic processes between ependymal cells to contact the CSF. This

primitive system serves a non-synaptic diffuse signal transmission function (Vigh et al.

2004).

The migratory phagocytic cells (supraependymal macrophages) are well defined.

They reside on the ventricular surface of ependymal cells. Migratory phagocytic cells

are descried as scavenger cells, but they might participate in immunological response

and iron regulation in the ventricular system or the brain as a whole (Ling et al. 1998).

Interactions with ependymal cells are not well understood, but it is known that

ependymal cells store factors (migration inhibitory factor (MIF), macrophage inhibitory

cytokine-1), produced by other cells (astrocytes, choroid plexus), which are capable of

regulating macrophage function.

26

2.2 Wnt Signaling system

In 1982, Nusse and Varmus identified a proto-oncogene Wnt1 (originally called

Int-1) as a signalling molecule affects the development of mammary tumors (Nusse et

al,. 1982). After several years, WNT1 and WG (Wingless), its Drosophila melanogaster

orthologue, emerged as key morphogens, which functions as regulators of the

embryonic body plan (Barker et al., 1988; McMahon et al., 1989). Wnts ligands are

secreted glycoproteins that play essential roles in embryogenesis and cortical

development. Wnts, as ligands, interact with 7-transmembrane, G-protein-coupled

receptors called Frizzleds (Fz) to initiate several different signaling pathways.

Figure 4. Wnt signaling pathways.

27

Canonical pathway (fig.4A) mediates gene induction events. This is the best-

characterized WNT signaling pathway, in which WNT binding to Frizzled receptors

activates the scaffolding protein Dishevelled (DVL), which via the DIX and PDZ

domains disassembles a so-called ‘destruction complex’ formed by glycogen synthase

kinase 3 (GSK3), Axin and adenomatous polyposis coli (APC) — a complex that

normally leads to the degradation of -catenin. WNT binding to Frizzled disrupts the

destruction complex, and this result in cytoplasmic stabilization of -catenin (He et al.,

2004; Liu et al., 1999) and its import into the nucleus, where it regulates gene

expression through association with lymphoid enhancer factor/T cell factor (LEF/TCF)

transcription factors. In this pathway, Frizzled collaborates with a co-receptor, LRP5/6

of the low-density lipoprotein receptor related protein (LRP) family.

The non-canonical or PCP pathway (fig.4B) mediates cell polarity, cell

movements during gastrulation and other processes, by signal transduction through the

PDZ and DEP domains of Dsh, leading to a modification of the actin cytoskeleton

(Weeman et al., 2003; Wallingford et al., 2002). At the level of Dsh, two independent

and parallel pathways lead to the activation of the small GTPases Rho and Rac.

Activation of Rho requires the formin-homology protein Daam1 that binds to the PDZ

domain of Dsh, leads to the activation of the Rho-associated kinase ROCK, and

mediates cytoskeletal re-organization (Habas et al. 2001; Marlow et al., 2002). Rac

activation is independent of Daam1, requires the DEP domain of Dsh, and stimulates

Jun kinase (JNK) activity (Habas et al., 2003; Boutros et al., 1998). Other Dsh-binding

molecules that influence the PCP pathway include Strabismus and Prickle, but their

mechanisms of action remain incompletely understood (Keller et al., 2002).

The Wnt-Ca2+ pathway (fig. 4C) is thought to influence both the canonical and

PCP pathways (Weeman et al., 2003). Wnt signaling through Frizzled receptors leads to

the release of intra-cellular Ca2+ in a process mediated through heterotrimeric G-

proteins and involving numerous other molecules, including phospholipase C (PLC),

calcium-calmodulin-dependent kinase 2 (CamK2) and protein kinase C (PKC) (Weeman

28

et al., 2003; Kuhl, 2002). The Wnt-Ca2+ pathway is important for cell adhesion and cell

movements during gastrulation.

2.2.1 Wnt biogenesis

Wnts are conserved in all metazoan animals. In mammals, complexity and

specificity in wnt signaling are achieved through Wnt ligands, which are cystein-rich

proteins of approximately 350-400 amino acids that contain an N-terminal signal

peptide for secretion. Murine Wnt 3a was the first purified and biochemically described

Wnt proteins (Willert et al., 2003). In addition to N-linked glycosylation, which is

reported to be required for Wnt 3a secretion (Komekado et al., 2007), Wnt 3a undergoes

two types of lipid modifications that likely account for hydrophobicity and poor

solubility of Wnt proteins (Hausmann et al. 2007). Willert and colleagues reported

lipidation for wnt ligands, it was addition of palmitate to cystein 77 (Willert et al. 2003).

Its mutation had minimal effect on Wnt 3a secretion, but diminished the ability of Wnt

3a to activate beta-catenin signaling pathaway (Galli et al., 2007; Komekado et al.,

2007; Willert et al. 2003). The second identified lipidation was a palmitoleoyl attached

to serine 209 and its mutation resulted in Wnt 3a accumulation in the endoplasmic

reticulum (ER) and a failure in secretion (Takada et al., 2006)

Two additional structures were identified for Wnt secretion: Wntless (Wls), also

known as Evenness interrupted (Evi) or Sprinter (Srt), in Drosophila, and the retromer

complex in nematodes (Hausmann et al., 2007). Wls is a multipass transmembrane

protein localized in the Golgi, edoplasmic compartments and in the plasma membrane.

Wls is essential for Wg/Wnt secretion. The retromer complex, which is composed form

five subunits was reported and defined first in the yeast. It mediates protein trafficking

between Golgi apparatus and endosomes (Hausmann et al., 2007). Loss of retromer

functions causes degradation of Wls in the lysosomes and results in reduction of Wls

and thus Wnt secretion

To conclude, Wnt is glycosylated and lipid modified by Porcupine in the ER and

is escorted by Wls from the Golgi to the plasma membrane for secretion. Wls is recycled

be endocytosis and trafficked back to Golgi by the retromer.

29

2.2.3 Wnt signalling and adult neurogenesis.

Adult neurogenesis in the mammalian brain is usually considered an active

process including the proliferation and cell fate specification of adult neural progenitors

(Duan et al., 2008). In the intact adult mammalian CNS, active neurogenesis occurs in

two ‘neurogenic’ regions: the subgranular zone (SGZ) of the dentate gyrus in the

hippocampus and the subventricular zone (SvZ) of the lateral ventricles in the forebrain

(Lie et al., 2004). New neurons are thought to originate from multipotent adult neural

stem cells, but their exact identity is still subject to debate and their multipotency at the

clonal level in vivo has not been universally demonstrated.

Recently, NEuROD1, a pro-neurogenic transcription factor in the adult brain that

is selectively expressed in dividing neural progenitors and in immature granule neurons

in the adult dentate gyrus, was identified as a downstream effector of Wnts in adult

neurogenesis (Kuwabara et al., 2009; Gao et al., 2009). WNT3A treatment induced the

expression of NEuROD1 in adult neural progenitors in vitro, and -catenin was directly

associated with the NEuROD1 gene promoter during the course of neurogenesis

(Kuwabara et al., 2009), Deletion of NEuROD1 in stem cells stopped neurogenesis in

vivo (Gao et al., 2009), and Wnt treatment of these cells did not stimulate neurogenesis

(Kuwabara et al., 2009). Wnt signalling has also been shown to modulate neurogenesis

in the SVZ. WNT3A and WNT5A increase the proliferation of cultured progenitor cells

dissected from postnatal and adult mouse SVZ and promote their neuronal

differentiation (Yu et al., 2006). In addition, retrovirus-mediated expression of stabilized

-catenin or treatment with an inhibitor of GSK3 promoted the proliferation of

progenitor cells in the SVZ. Conversely, expression of the Wnt antagonist DKK1

reduced the proliferation of progenitor cells (Adachi et al., 2007). These studies indicate

that activation of Wnt signaling regulates adult neurogenesis in the SVZ by regulating

progenitor cell proliferation.

Stem cell differentiation and proliferation are controlled by both intrinsic and

extrinsic regulators. Wnt ligands are among the extracellular factors that affect this

process (Ming et al., 2005; Nusse, 2008). During development Wnts act on CNS

progenitor cells, and the activation of -catenin leads to the proliferation of the neural

30

progenitors, resulting in the expansion of the entire neural tube (Chenn et al., 2002). In

addition, a GSK3 inhibitor was reported to induce the selective differentiation of stem

cells into neurons, and WNT7A promoted the maturation of neural precursor cells into

mature neurons (Hirabayashi et al., 2004. Wnts secreted by hippocampal progenitors

self-stimulate canonical Wnt signalling, and inhibition of this autocrine Wnt pathway

increases the number of neurons and leads to a loss of the multipotency of the

progenitors (Wexler et al., 2009). Inhibition of Wnt signalling by lentiviral expression of

a dominant negative Wnt in the dentate gyrus reduces neurogenesis in the hippocampus

(Zhou et al., 2002) and decreases long-term retention of object recognition in adult rats

(Jessberger et al., 2009)

2.2.4 Wnt signalling and Alzheimer disease (AD)

Studies in humans indicate that Wnt signalling is related to neurogenesis and is

altered or involved in the pathophysiology of AD. An example is the reduced renewal

capacity of glial-like progenitor cells dissected from the temporal cortex of patients with

AD — which correlated with elevated levels of GSK3 activity and increased

phosphorylation of -catenin — compared with that of cells from healthy controls (He et

al., 2009). Moreover, treating glial precursor cells from healthy brains with amyloid-

peptide (A) also led to increased -catenin phosphorylation level and reduced

neurogenesis. Conversely, -catenin transfection led to restoration of neurogenesis (De

Ferrari et al., 2000). These studies suggest that Wnt signaling is required for human

cortical neurogenesis, impaired Wnt signalling reduce the capacity of progenitors to

undergo neurogenesis and contribute to repair.

Almost a decade ago a relationship between loss of Wnt signalling and A

induced neurotoxicity was proposed (De Ferrari et al., 2000, De Ferrari et al., 2003).

The alteration in Wnt signalling was suggested to be the triggering factor for A

production and tau hyperphosphorylation, which induce synapse and neuron loss (De

Ferrari et al., 2003; Mudher et al.,2002). Since then, other studies have shown that

several Wnt signalling elements are altered in AD (Mudher et al., 2002; Caricasole et

al., 2003; Smal et al., 2008; Boonen et at., 2009). A directly binds to the Fz5 receptor

cysteine-rich domain at or in close proximity to the Wnt-binding place, inhibiting the

31

canonical Wnt signalling pathway (Magdesian et al., 2008). In addition, genetic studies

demonstrated a link between Wnt signaling and AD. The apolipoprotein E �4 allele,

which is supposedly associated with an increased risk of developing AD, inhibits

canonical Wnt signalling on stimulation with WNT7A (Caruso et al., 2006).

To find out whether activation of Wnt signaling could protect hippocampal

neurons from the A toxicity, the effects of activating other signalling pathways that

crosstalk with the Wnt signaling pathway was examined (Inestrosa et al., 2008). As

cholinergic dysfunction has been observed in patients with AD, treatment rodents with

an M1 muscarinic AChR receptor agonist (Farias et al., 2004) or nicotine (Inestrosa et

al., 2008) was examined. Activation of Wnt signalling through cholinergic activation

seems to be a neuroprotective mechanism against A. In fact, it is well known that M1

agonists increase the non-amyloidogenic processing of the amyloid precursor protein

(APP), reducing A production and toxicity. In addition, cholinergic activation by the

specific M1 agonist induces the phosphorylation of GSK3 in neuronal cultures from

transgenic mice that overexpress GSK3102. Ser9 phosphorylation of GSK3 by

cholinergic stimulation is probably mediated by a mechanism involving protein kinase C

(PKC), as it was blocked by a PKC inhibitor (Farias et al., 2004). The protection

observed in vitro has been confirmed in vivo. Treatment with the specific M1 agonist

improved the spatial memory and reduced the A load in the hippocampus of transgenic

mouse model of AD. These results show that cholinergic activation interacts with the

Wnt signalling pathway, leading to potential neuroprotection against A toxicity.

Recent evidence suggests that lithium is neuroprotective agent in various

neurodegenerative diseases and it it should be noted that lithium reduces the prevalence

of AD in elderly patients with bipolar disorders (Nunes et al., 2007). Studies in a mouse

model of AD indicated that lithium reduces the size of the amyloid burden, including the

A oligomers, and prevents the behavioural alterations of the animals (Toledo et al.,

2009). Under these conditions lithium activates Wnt signalling, by the inhibition of

GSK3 and the increase in -catenin (Toledo et al., 2009). These studies are consistent

with the hypothesis that loss of Wnt signalling is involved in A-dependent

neurodegeneration and activation of the canonical Wnt pathway by lithium protects

against the synaptic changes triggered in AD.

32

The known functions of Wnt signalling in the adult brain suggest that disruptions

or impairments of the Wnt pathways could have dramatic consequences. Indeed,

growing evidence implicates Wnt signaling in the pathogenesis of several

neurodegenerative and neurological diseases. In the future it will be important further

explore the molecular mechanisms that link these pathways to disease, in particular to

the pathogenesis of Alzheimer disease and Parkison disease.

The emerging roles of Wnts in adult neurogenesis, neuronal differentiation,

synaptogenesis and survival suggest that targeting Wnt signalling pathways could offer

therapeutic benefits. Drugs capable of modulating Wnt signalling may become tools for

regenerative or neuroprotective medicine, for example against diseases associated with

neuron loss.

2.2.5 The structure of Dvl

Dvl proteins have three conserved domains: an aminoterminal DIX domain of 80

amino acids, a central PDZ domain of about 90 amino acids, and a carboxyl-terminal

DEP domain of 80 amino acids (Boutros et al., 1999). In addition, another two

conserved regions, the basic region and the proline-rich region, are also implicated to

mediate protein–protein interaction and/or phosphorylation.

The DIX domain. Both overexpressed and endogenous Dvl proteins form

cytoplasmic puncta (Yanagawa et al., 1995; Yang-Snyder et al. 1996). Recent studies

Figure 5. Dvl domain structure. Dvl protein is about 700 amino acids, harboring conserved DIX, basic and serine/threonine-rich region, PDZ, praline-rich region and DEP domains.

33

revealed that Dvl puncta undergo dynamic polymerization, which to some extent

correlate with the ability of Dvl to activate Wnt/-catenin signaling (Schwarz-Romond

et al., 2005; Smalley et al., 2005). Dvl polymerization is mediated by its DIX domain

(Wharton et al., 2003). The structure of Dvl's DIX domain has not been defined, but the

crystal structure of DIX domain of rat Axin, which is closely related and shares similar

properties of Dvl's DIX domain (Schwarz-Romond et al., 2007; Fagotto, et al. 1999),

has been solved (Schwarz-Romond et al., 2007). The single DIX domain has a compact

fold with five -strands and one -helix. The structure reveals that multiple -strands

engage in head-to-tail interaction between two different surfaces of DIX domains. Some

of the residues in the core structure or those involved in the 2–4 interactions of the

Axin DIX domain are highly conserved in the DIX domains of Dvl. Mutations at the

core structure or the interaction surface strongly diminish the Wnt signaling (Schwarz-

Romond et al., 2007).

The PDZ domain. PDZ domain is a modular protein interaction domain that has

tow helices and six sheets, which together with the preceding loop form a peptide-

binding cleft (Cheyette et al.. 2002). Dvl exploits its PDZ domain to transducer signals

from the membrane receptor Fz to downstream components by direct interaction with Fz

(Wong et al., 2003; Barker and Clevers, 2006). The special role of the Dvl PDZ domain

in the Wnt pathway makes it an ideal pharmaceutical target (Barker and Clevers, 2006).

The therapeutic usefulness of PDZ protein–protein interaction interference has been

clearly shown using small peptide (Chen et al., 2009; Lee et al., 2009; Zhang et al.,

2009) or non-peptide antagonists which block the PDZ-mediated interactions (Fujii et

al., 2007; Grandy et al., 2009). For example, a virtual screen of small molecule

compounds in the three-dimensional databases has identified molecules predicted to

bind to the Dvl PDZ domain and therefore inhibit its interaction with Fz (Shan et al.,

2005). At least two promising candidates, NSC668036 (Shan et al., 2005) and

compound 3289–8625 (Grandy et al., 2009), have been subsequently synthesized and

tested for their capacity to interact with Dvl using NMR spectroscopy.

The DEP domain. The DEP domain in the C-terminal region of Dvl consists of

a bundle of three -helices, a -hairpin “arm” composed of two -strands, and two short

34

-strands. In light of the structure, the protein surface that includes K434, D445, and

D448, is predicted to be crucial for the interaction between Dvl and other proteins and to

play an important role in signal transduction (Wong et al., 2000) It was recently

suggested that the basic residues in the DEP domain are essential for the binding.

The basic region and Pro-rich region Preceding the amino terminal of the PDZ

domain is a cluster of positive charged (basic) residues. This region contains the

phosphorylation sites targeted by CK2 and PAR-1 (Willert et al. 1997), and together

with the PDZ domain, this region is involved in the direct binding with the EFX domain

of Naked Cuticle (Nkd) (Rousset et al. 2001).

2.2.6 Dishevelled controls apical docking of basal bodies and planar cell

polarization.

The motile cilia of multi-ciliated cells are very significant to the physiology of a

variety of organs. There are accumulating findings demonstrating Dvl involvement in

the formation of basal bodies. First, Dvl is required for actin erection and basal body

docking to the apical cell surface of ciliated cells. Dvl might be required for

maintenance of basal bodies at the apical surface. Dvl with Inturned, the effector

protein, are required for local activation of the Rho GTPase, specifically at the apical

surface of ciliated epithelial cells. It is already reported that Dvl links basal body

docking to vesicle traffic and Sec8 localization (Park et al., 2008).

Transmission Electron Microscopy (TEM) studies of both multi-ciliated cells

and cells with only a primary cilium have suggested that basal body docking is achieved

by association of basal bodies with cytoplasmic vesicles, which then fuse to the apical

cell surface by a mechanism similar to exocytosis (Sorokin et al., 1968; Cohen et al.,

1998). It is possible then to predict a model whereby Dvl associates closely with the

basal body complex and the recruitment of Sec8-positive exocytic vesicles. Inturned

may serve as a scaffold that places Rho closely with Dvl, thereby activating the GTPase

(Habas et al., 2001; Park et al., 2005). Because actin is crucial for fusion of vesicles to

the plasma membrane (Lanzetti, et al. 2007), it is noteworthy that apical actin assembly

is also altered in multi-ciliated cells lacking Dvl or Inturned. Dvl probably controls actin

35

assembly in multi-ciliated cells via CapZIP, which is phosphorylated downstream of

Dishevelled (Oishi et al., 2006).

Although the detailed mechanisms of Dvl function in basal body docking remain

to be covered, this link to vesicle traffic has broad implications. In addition to basal

body docking, Dvl controls canonical Wnt signaling and PCP signaling, but a unifying

mechanism for Dvl function has yet to be find out (Wallingford et al., 2006). An

association between Dvl and membranous vesicles is a matter of persistant debate

(Schwarz-Romond et al., 2005); however, several lines of evidence support this

hypothesis. First, association of Dvl with vesicles has been shown to be important for

canonical Wnt signaling (Capelluto et al., 2002), and it administers the aggregation of

the Wnt co-receptor Lrp6 into membrane-associated signalsomes (Bilic et al., 2007).

Second, intracellular PCP signaling components, including Dvl, interact functionally

and physically with elements of the membrane trafficking machinery (Yu et al., 2007;

Classen et al., 2005; Kishida et al., 2007; Chen et al., 2003). Finally, Dvl has been

included in endo- and exocytosis at synapses (Ahmad-Annuar 2006; Kishida et al.,

2007).

A connection between Dvl, membrane traffic and the basal body is especially

intriguing in light of recent results linking cilia and Wnt signaling. The cilium and basal

body are necessary organelles governing the activity of Wnt signaling pathways

(Simons et al. 2005; Ross et al., 2005; Gerdes et al., 2007). The interconnections

between Wnt signaling, vesicle traffic and ciliogenesis are best explained by the BBS4

protein. BBS4 cooperates genetically with both canonical Wnt and PCP signaling (Ross

et al., 2005; Gerdes et al., 2007), but it also mediates membrane delivery to the primary

cilium and controls microtubule organization and ciliogenesis (Kim et al., 2004;

Nachury et al., 2007). Moreover, ciliary and basal body proteins can directly regulate by

Dvl phosphorylation, localization and stability (Gerdes et al., 2007; Oishi et al., 2006).

Connecting these disparate findings will be a challenge for future investigation.

It is also important that both Dvl and Rho play roles in apical docking that are

experimentally separable from those involved in planar polarization of basal bodies. The

direction of ciliary beating is formed subsequent to basal body docking and cilia

outgrowth (Boisvieux-Ulrich et al., 1985; Frisch et al., 1968; Mitchell et al., 2007), it

seems reasonable to conclude that Dvl and Rho act first in apical docking and only later

36

affects planar polarization. Nonetheless, it will be important to find out how Dvl at the

basal body discriminates between its apical docking function and its planar polarity

function. One possibility is that Rho activity for apical docking and for planar

polarization regulated by distinct guaninenucleotide exchange molecules. This idea is

supported by the evidence that knockdown of ARHGEF11 causes a spectrum of

embryonic defects, resulting defective cilia-mediated fluid flow (Panizzi et al., 2007;

Kramer-Zucker et al., 2005). In ARHGEF11 morphants, only apical actin is disrupted in

multi-ciliated cells, but cilia are grossly normal and motile (Panizzi et al., 2007), so this

exchange factor may function exclusively in polarization of ciliary beat.

37

3. Experimental part 3.1 Materials and methods 3.1.1 Cell culture

The preparation of culture mediums and all manipulations of cells including

cell isolation and treatments were carried out under a laminar flux under sterile

conditions. For immunocytochemistry cells were culturedonto coverslips in 35-mm Petri

dishes.

Cells isolation from rat SVZ. SVZ cell cultures were prepared form newborn

rats, which were scarified at PO or P1 by decapitation. Brains were removed from the

skull and put in one drop cold Hank’s solution in a 100 mm Petri dish. By visualizing

with microscope first coronal cut was performed in order to remove the rostral part of

the hemispheres. A second coronal cut was performed at the level of anterior horn of

lateral ventricles. The ependymal cells were quickly removed with forceps at the third

ventricle and put in a 100 mm Petri dish with cold Hanks solution to preserve the tissue.

Once finished all animals SVZ cells were add in 3 ml Hanks into using and very gently

homogenized using 1 ml pipette. Then the tube was filled up to 10ml and trypsin (10

µm/brain) was added. Cells were incubated in tripsin for 15 minutes at 37ºC in order to

���������������� ����� �������������

�������������� ������������������ ��

Figure 6. Isolation of Ependymal cells. Ependymal cells were dissected from third ventricles of PO-P1 rat puppies. It later was cultured on the laminin coated dish.

38

digest intracellular contacts and separate cells. Then, tripsin was stopped with 1 ml FCS

and cells recovered be centrifugation at 1200rpm/min for 10 minutes. Cells were

resuspended in 1 ml complemented Neurobasal medium (Gibco Brl, Paisley, Uk) and

washd three times in Neurobasal medium. Cells were counted, resuspended in medium

an homogenously mixed by using a vortexing machine before plating them onto laminin

coated cell culture supports. Cells were allowed to grow in Neurobasal medium with 2

% B27 supplement, 2mM glutamaine and 1 mM sodium pyruvate at 37 ºC and 5% CO2.

Cells were cultured for 7-10 days and medium was replaced 2 times during that period.

This ependymal cells culture consisted of atrocities, neuroblasts, neurons and

ependymal cells. Ependymal cells were at the different level of maturation.

Immunostaining on ependymal cell culture. In the begging cells were with 2

ml of PAF 4% for 30 minutes. In order to remove remaining PAF 4% it was perform a

3 times washing with PBS for 10 minutes. PBS solution was removed and added 2ml

PBS-BSA-Tryton solution (for 1h) to cover unspecific antigens. Next step was to add

100µm primary antibody (Wnt 3a, 5a, 7a, 7b, 8b and Dvl 1, 3 were diluted 1:50 in PBS -

0.5%; Dvl-2 was diluted 1:100 in PBS -0.5% ) and laeve overnight at 4ºC temperature.

After ~12h all volume of primary antibody was removed and there were three series of

washing with PBS. Later secondary fluorescence antibody (diluted 1:1000 into PBS-

BSA) was added on the cells and kept it for 1h at room temperature (without light).

Solution with antibody must be removed and again washed three times with PBS. In

order to stain nucleus, I used Hoechst fluorescent DNA-binding compound. Prepared

solution (1:5000 in PBS) was deposited on the slices for 30 minutes at room

temperature. In the end slices were mounted: one drop of PBS- glycerine on the cells

and cover with slice. Slides were dried at room temperature.

During each immunolabeling experiment a negative-control was done, where

only the secondary antibody was added to the cells; in this way the background intensity

provoked by unspecific bound could be assessed.

39

Fixation. The step of fixation plays an essential role to preserve the cellular

structure and to avoid the risqué of morphological modifications. In general our cells

were fixed with paraformaldehyde solution at 4 % diluted in phosphate buffer. This

chemical fixator is the polymerized form of formaldehyde, reacting especially with

amino groups of the proteins thus creating bridges between between different

polypeptide chains. It ensures good preservation of nucleoproteins and proteins.

Sometimes this fixative preserves the enzymatic activity but never the lipids

constituents.

Specimens were carefully transferred into the dish filled with 4% PAF for an

overnight fixation at 4°C (cell culture were fixed just for 30 minutes)

3.1.2 Brain slices

Vibratome section’s preparation. Rats were anesthetized with sodium

pentobarbital (50mg/kg, intraperitoneally) and perfused through the ascending aorta

with saline rinse, followed by 500ml of PAF 4% fixative. After removal, brains were

placed in the fixation solution overnight and block for Vibratome sectioning the

following day. Tissue sections were cut into PBS buffer on Vibratome at 50 µm. Tissue

specimens are ready for immunostaining

Immunostaining on vibratome sections. The most suitable for experiment

tissue specimens were selected. Slices were separated 2-6 per well. In order to remove

remaining PAF 4% it was perform a 3 times washing with PBS for 10 minutes. PBS

solution was removed and added PBS-BSA-Tryton solution (for 1h) to cover unspecific

antigens. After removing previous solutions, primary antibody (Wnt 3a, 5a, 7a, 7b, 8b

and Dvl 1, 3 were diluted 1:50 in PBS -0.5%; Dvl-2 was diluted 1:100 in PBS -0.5% )

was added and left it for 48 hour at 4ºC temperature. After 48h all previous solution

were removed and there were three series of washing with PBS. Later secondary

fluorescence antibody (diluted 1:1000 into PBS-BSA) was added on the slices and kept

it for 2h at room temperature (without light). Solution with antibody must be removed

and again washed three times with PBS. In order to stain nucleus, I used Hoechst

40

fluorescent DNA-binding compound. Prepared solution (1:5000 in PBS) was deposited

on the slices for 30 minutes at room temperature. In the end slices were mounted: one

drop of PBS- glycerine on the sections and cover with slice. Slides were dried at room

temperature.

During each immunolabeling experiment a negative-control was done, where

only the secondary antibody was added to the cells; in this way the background intensity

provoked by unspecific bound could be assessed.

Wholemount Dissection. The animal was sacrificed by cervical dislocation

and the head is cut off. A midline incision is made, posterior to anterior, along the scalp

to reveal the skull. A series of 4 cuts in the skull are made to open the cranium: one cut

spans the two orbits anterior to the olfactory bulbs, the next two cuts are inferior to the

cerebellum and separate the cranium from the skull base and the final cut runs posterior

to anterior along the mid-sagittal suture. The cranial flaps are gently retracted and the

brain is extracted and placed into the dissecting dish. The dissection is performed under

the stereomicroscope. First, the olfactory bulbs are dissected away from the brain.

Divide the brain along the interhemispheric fissure. A coronally-oriented cut is then

made at the posterior-most aspect of the interhemispheric fissure, allowing the caudal

hippocampus to be visualized in cross-section. The hippocampus, which forms the

medial wall of the lateral ventricle at this position, must then be released from the

overlying cortex, which forms the dorsal-lateral wall of the ventricle. First, the knife is

inserted into the small ventricular space between the cortex and hippocampus dorsally,

and a cut is made in the cortex where it reflects ventrally, away from the midline, to join

the hippocampus. After this cut is made, the cortex can be slowly peeled away from the

hippocampus to reveal the lateral ventricle moving from dorsal to ventral. This

maneuver is expedited by cutting off a wedge of cortex at the corner where the

hippocampus was released. After reaching the ventral-most extent of the lateral ventricle

at this position, you may either visualize or feel where the cortex again wraps around,

this time reflecting back medially, to join the hippocampus. Another cut must be made

in this position to completely release the hippocampus or medial wall of the lateral

ventricle from the cortex or lateral wall of the lateral ventricle. The hippocampus is

pulled away from the cortex, medially and anteriorly, to open the lateral ventricle

41

widely. The hippocampus is pulled anteriorly using small strokes of the forceps and

knife to retract the medial and lateral walls apart. Once the resistance to this retraction

begins to increase, additional cuts are needed. First, to increase your exposure to the

lateral ventricle and in particular, the lateral wall and SVZ, dissect away the cortex. The

cortex is cleanly dissected away by visualizing the interface between the corpus

callosum and the VZ/SVZ. Simply cut along this interface staying on the callosal side to

avoid damaging the SVZ. In order to continue retracting the medial wall away from the

lateral wall, two more cuts are needed: one cut dorsally where the lateral wall, medial

wall, and cortex all converge, and one cut ventrally where the lateral wall, medial wall,

and thalamus converge. With these cuts made, further gentle retraction on the medial

wall allows the anterior-most extent of the lateral ventricle to be opened. Next step is to

separate the medial and lateral walls anteriorly. Cut exactly in this valley to separate

these two walls. The specimen is ready for further investigations.

Cryostat section’s preparation. Wholemount was obtain by following steps

as described above and overnight fixed with PAF 4%. Fixed wholemount specimen was

saturated in cryoprotectant (30% sucrose in 0.1M PBS for 48h).

Evenly place embedding solution on base of chilled platform. As the solution

begins to freeze, carefully align and place tissue specimen (brain or wholemount,

ventricle side down) on platform. The wholemount should adhere to the platform. Cover

entire specimen with embedding solution. This may take several application/freezing

steps to accomplish. Set the cutting temperature of cryostat to -20ºC. Section brain in

cryostat at a thickness of 20 µm. Each section were transferred to a subbed slide. Slides

were placed on warming plate at 40ºC to dry and put in holder, then freezed at -20ºC

overnight. Tissue specimens are ready for immunostaining

Immunostaining on cryostat sections. Slides with sections were defreezed at

room temperature for 1.5h. Later, they were kept in PAF 4% for 1h. In order to remove

tissue-teck and PAF 4% slides were rinsed 3 times with PBS for 10 minutes. Next step

is to paint with special pen around the sections and add the solution PBS-BSA-TX (to

cover unspecific sites) for 30 minutes. After removing previous solutions, primary anti-

Wnt 8b antibody (1:50 diluted in PBS -0.5%) was added and left it for 48 hour at 4ºC

42

temperature. After 48h all previous solution were removed and there were three series of

washing with PBS. Later secondary fluorescence antibody (diluted 1:1000 into PBS-

BSA) was added on the slices and kept it for 2h at room temperature (without light).

Solution with antibody must be removed and again washed three times with PBS. In

order to stain nucleus, I used Hoechst fluorescent DNA-binding compound. Prepared

solution (1:5000 in PBS) was deposited on the slices for 30 minutes at room

temperature. The last step is mounting the slice: several drops of the PBS- glycerine on

the sections and cover with slice. Let the slide dry at room temperature (without light)

and it is ready for analysis.

3.1.3 Analysis

Fluorescent images were obtained with a confocal microscope (Zeiss LSM 510 Meta,

Carl Zeiss Inc., Thornwood, NY), and digital images were captured and imported with

the LSM 5 image browser (Carl Zeiss Inc.).

43

Figure 7. Immunohistochemical analysis of Wnt 8b. A – Intensive signal in the ependymal cells (scale bar 100 µm). B – Clusters of cilia are labeled, no staining in ependymal cells bodies (scale bar 20µm). C – confocal analysis reveals Wnt 8b immunoreactivity just in basal part of the cilia (scale bar 5 µm). Red –Wnt8b, green – tubulin

3.2 Results

3.2.1 Analysis of Wnt ligands.

Wnt signaling proteins affects many aspects of animal development and

regeneration after injury. Wnt8b has been reported to be expressed in the dorsomedial

telencephalon, hippocampus and other brain regions (Tole et al., 2000; Theil et al.,

2002; Kimura et al., 2005; Shimogori et al., 2004), but immunohistochemical analysis

in the ventricular zone cells (ependymal) has not been done before.

Immunohistochemical experiments with WNT 8b antibody were carried out on

coronal vibratome sections from perfused and fixed adult rat brains. Low power analysis

with a fluorescence microscope of all brain structure revealed strong immunoreactivity

in the subventricular zone of third (Figure 7. A) and lateral ventricles. Also, we detected

strong signal of Wnt 8b in the hippocampus, these results corresponds to Lako and

colleagues findings in the mouse brains (Lako et al., 1998). Analysis with a high power

confocal microscope helped to detect immunoreactivity on the apical surface of

ependymal cells (Figure 7. B)

44

Figure 8. A – confocal image of “ whole mount material” , confirms wnt 8b accumulation in the basal part of the cilia (In red – Wnt 8b, green – tubulin, scale bar 10µm)

In order to localize Wnt 8b signal in the

ependymal cells, I used acetylated tubulin antibody that

labels stable microtubules (Bulinski et al., 1988) within

the cilia. Double labeling for acetylated tubulin and

Wnt 8b reveals that high levels of Wnt 8b appear to

localize in the basal part of the cilia (Figure 7. C)

Analysis of negative control (than primary wnt8

antibody is emitted) did not show any immunostaining

In order to confirm these results of wnt8b

staining, whole mount preparation protocol was applied

to prepare brains. Immunostaining was done on brain

sections cut with cryostat. Results of this experiment

were similar to the conventional immunohistochemical

protocol (Figure 8. A). The immunoreactivity pattern of Wnt 8b antibody, including

results with whole mount protocol, is very similar in ependymal cells lined all ventricular

system of adult rat’ s brains.

Further, to find out whether WNT 8b immunoreactivity is specific, I have

conducted several immunostaining experiments with other Wnt ligand’ s antibodies: Wnt

Figure 9. Low magnification pictures were taken in the lateral ventricles. Wnt 5a (A), Wnt 7a (B), Wnt 7b (C) – show diffuse, unspecific, very weak signal in the ependymal cells. Scale bars in all pictures are 100 µm)

45

3a, Wnt 5a, Wnt 7a and Wnt 7b. Sections for this experiment were prepared using the

same conditions as in the beginning – perfused and fixed adult rat brain was cut coronally

with vibratome. Analysis of experimental material with fluorescence and confocal

microscope did not show any specific staining of ependymal cells (Figure 9. A-C). Wnt 3a

did not show any staining.

To do further analysis, I have established ependymal cells culture. Third

ventricle’ s ependyma layer was dissected from PO – P1 rats brains and cells were cultured

in the dish. Cells fixation and immunostaining were done after 7 -10 days of culturing.

Analysis with fluorescence and confocl microscope revealed intensive signal in the

ependymal cells (Figure 10. A,B). Wnt 8b antibodies mostly labeled the cilia of the

ependymal cells, but not their cell body. Also, double staining was done with Wnt 8b and

acetylated tubulin (it labels all the length of the cilia). This experiment determined that

Wnt 8b is accumulated just in the basal part of the cilia of the ependymal cells (Figure 10.

C). Negative control (than primary wnt8 antibody is emitted) and experiments with Wnt

3a, Wnt 5a, Wnt 7a and Wnt 7b antibodies showed no signal in the ependymal cells.

Figure 10. Immunostaining results of cultured ependymal cells. Most of the Wnt 8b is accumulated in the cilia (A – scale bar 10µm; red – wnt 8b), but not in the cell body of ependymal cells (B – scale bar 10µm; green - tubulin). Cofocal analysis indicates the strongest immunoreactivity just in the basal part of ependymal cells (C – picture from side; in green – tubulin, red – Wnt 8b).

46

Figure 11. A – Low magnification picture of Dvl-1 in the lateral ventricle. Ependymal cells are highly labeled, lateral and dorsal walls are more positive, than medial one (scale bar is 100 µm). B – picture of lateral wall of lateral ventricle. Staining of ependymal cells is not homogeneous; signal in the nucleus is intensive (scale bar is 20 µm). C - Low magnification picture of Dvl-2 in the lateral ventricle. There is no specific staining in the ependymal cells.

3.2.2 Analysis of Wnt signal transducer - Dishevelled

The phosphoprotein Dishevelled (Dvl) is an essential component of Wnt signaling

pathways and transduces signals into three separate branches: the canonical, non-

canonical and Ca2+ pathways (Miller et al., 199; Logan et al., 2004). In all three pathways

Dvl operates at the plasma membrane or in the cytoplasm. Numerous of studies are done

with Dvl isoforms, and for example it is known that Dvl1 is highly expressed in

hippocampus (Krylova et al., 200) or in areas of high neuronal density at embryonic and

postnatal stages of development (Sussman et al., 1994). I was unable to find in the

literature any studies done concerning ependymal cells and all dishevelled isoforms.

To examine all Dvls in ependymal cells, I have conducted immunohistochemical

experiments on coronal vibratome sections from perfused and fixed adult rat brains. Low

power analysis with fluorescence microscope revealed intensive immunoreactivity of Dvl-

1 antibody in the lining of the ventricular system. Staining is not homogenous, random

cells are more positive. Also, there are differences between walls of the lateral ventricles:

lateral and dorsal walls are labeled stronger than medial one (Figure 11. A). In order to

determine cellular localization of Dvl-1 in ependymal cells, I have applied confocal

microscope. This unraveled that Dvl-1 mainly is accumulated in the nucleus of the cells

(Figure 11. B). Negative experiment, conducted after excluding primary antibody showed

no immunoreactivity in the ependyma.

47

Figure 12. A - Low magnification picture of Dvl-3 in the lateral ventricle. Intensive signal is in the ependymal cells; dorsal and lateral walls are labeled more positive than medial one (scale bar 100 µm). B – high magnification picture of lateral wall of lateral ventricle. Staining in ependymal cells is much stronger than in surrounding layers. Dvl-3 antibody labeled membrane and cytoplasm of ependymal cells (Scale bar 20 µm). C - Confocal image, clearly confirms that Dvl-3 in ependymal cells is accumulated close to the membrane, the concentration in the cytoplasma and nucleus is low.

The same experimental procedures, as mentioned above, were applied to

investigate Dvl-2. Analysis with fluorescence and confocal microscope detected no

immunoreactivity in the ependymal cells lined all ventricular system of the brains (Figure

10. C).

The same procedure as for Dvl-1 was used for the exploration of Dvl-3 in the

ependymal cells. Firstly, low power analysis show strong labeling of ependymal cells.

Immunoreactivity appears to be more intensive in the ependymal cells lined lateral and

dorsal walls of the lateral ventricles compare to medial wall (Figure 12. A, B). Confocal

analysis exposed intensive membranous staining of the ependymal cells (Figure 12. C).

Negative control (with omitted primary antibody) did not possess any labeling.

To do further analysis with Dvls, I have used already establish ependymal cell

culture. Confocal analysis of immunohistochemical experiments with Dvl-1 antibody

confirmed, that this prorein is mostly accumulated in nucleus (Figure 13. A). Stronger

nuclear labeling can be observed in ciliated ependymal cells comapared to not ciliated.

Double labeling for Dvl-1 and acetylated tubulin reveals that some of the Dvl-1 is

accumulated in the basal part of the cilia (Figure 13. B,C). Dvl-1 immunoreactivity in the

cilia compared to Wnt 8b is less intensive.

DVL-3 C

48

Immunostaining on cultured ependymal cells with Dvl-3 antibody confirmed,

previously observed, stronger signal in the cytoplasm compared to nucleus. High power

analysis with confocal microscope exposed membranous staining of mature ependymal

cells (which are clustered together) (Figure 13. F). There are no cilia staining. There is

no specific staining of Dvl-2 in cultured ependymal cells (Figure 13. D,E).

All Immunohistochemical experiments are controlled by negative control (with

omitted primary antibody), to ensure, that primary antibody is specific. In order to find

Figure 13. Results of immunohistochemical experiments with cultured ependymal cells. A - white arrows indicate strongly labeled nucleus by Dvl-1 antibody (scale bar 10µm). B – Dvl-1 is also accumulated in the cilia of ependymal cells (ependymal cell body is not visible very well, because it is focused on cilia; scale bar 10µm). C – immunostaining of acetylated tubulin antibody. It shows the same place as in picture B. Cilia, marked by tubulin, are in the same place as structure labeled by Dvl-1 (B) antibody ( green –acetylated tubulin; scale bar 10µm). D – Dvl-2 labeling is not specific and very weak (sacle bar 10µm). E – immonuhistochemical experiment with acetylated tubulin. Picture were taken at exact place as D in order to confirm that Dvl-2 do not label even mature and very ciliated ependymal cells (in green – acetylated tubulin; scale bar 10µm). F – experiment with Dvl-3 antibody. The membrane of ependymal cells is labeled, also weak labeling of cytoplasm.

49

the best immunohistochemical protocol to work with brain’ s slices, antigen retrieval

protocol was performed to all antibodies. There was no significant difference observed

between conventional and antigen retrieval protocols. Experiments were repeated several

times and different animal’ s brains were used.

Table 1. Summary of the results.

Wnt3A

Wnt5A

Wnt7A

Wnt7B

Wnt8B

Dvl-1

Dvl-2

Dvl-3

Cultured rat ependymal cells: • Cilia

• Cytoplasm

• Nucleus

• Membrane

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

+++

-

-

-

++

+

+++

-

+/-

+/-

+/-

+/-

-

++

+/-

++

Rat brain slices: • Cilia

• Cytoplasm

• Nucleus

• Membrane

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

+++

-

-

-

++

+/-

+++

-

-

-

-

-

-

++

-

+++

- - no signal;

+/- - very weak or uspecific signal;

+ - weak signal;

++ - good signal;

+++ - Strong signal.

50

3.3 Discussion

WNT ligands are secreted proteins that regulate cell fate decisions, cell polarity, cell

migration, axonal morphology, and synaptic differentiation (Lucas and Salinas, 1997;

Wodarz and Nusse, 1998; Hall et al., 2000; Patapoutian and Reichardt, 2000). In

mammals, complexity and specificity of Wnt signaling are in part achieved through 19

Wnt ligands (http://www.stanford.edu/~rnusse/wntwindow.html). Several studies reported

that Wnt genes continue to be expressed in the adult brain (Parr et al. 1993) and possess

significant functions, like Wnt 5a through the non-canonical pathway regulate the

development of midbrain dopaminergic neurons (Castelo-Branco et al., 2003) or Wnt7a

affects axon guidance and remodeling (Hall et al., 2000). With that in mind, I have done

immunostaining with antibodies for Wnt 3A, Wnt 5A, Wnt 7A, Wnt 7B and Wnt 8b,

previously reported in several brain regions. Analysis of Wnt 8b immunohistochemical

experiment revealed very intensive immunoreactivity in the basal part of ependymal cell’ s

cilia. This is a novel localization of Wnt 8b in ependymal cells. There was no staining

with with Wnt 3a, Wnt 5a, Wnt 7a and Wnt 7b antibodies in vivo and in vitro. These

results suggest that staining with Wnt8b antibody in ependymal cells is specific.

The phosphoprotein Dishevelled (Dvl) is an essential component of Wnt signaling

pathways and transduces signals into three separate branches: the canonical, non-

canonical and Ca2+ pathways (Miller et al., 199; Logan et al., 2004). In all three pathways

Dvl operates at the plasma membrane or in the cytoplasm. Numerous of studies are done

with Dvls isoforms, and for example it is known that Dvl1 is highly expressed in

hippocampus (Krylova et al., 200) or in areas of high neuronal density at embryonic and

postnatal stages of development (Sussman et al., 1994). During review of the literature, I

was unable to find any immunohistochemical studies done concerning ependymal cells

and all dishevelled isoforms. Therefore, these are new results: Dvl-1 can be detected in the

nucleus and in the cilia; Dvl-3 is mostly accumulated in the cytoplasm and on the

membrane; Dvl-2 showed no specific staining.

51

3.3.1 Ependymal cells can secrete Wnt 8b

How are wnt proteins secreted? In Wnt-producing cells, the Wnt protein becomes

palmitoylated at two sites in the endoplasmic reticulum by the porcupine acyl-transferase.

The exact role of lipid modification remains unclear. A possible function is the targeting

of Wnts to particular domains of the membrane. Further transport and secretion of the

Wnt protein in secretory vesicles is controlled by the multi-pass transmembrane protein

Wntless/Evi (Ching et al., 2006), which is present in the Golgi and/or on the plasma

membrane. The mammalian ortholog of Drosophila Wntless/Evi/Sprinter is GPR177. The

retromer complex, including VPS35, may act within Wnt-producing cells to generate Wnt

forms that can be transported outside cells, possibly in the form of a lipoprotein particle.

It is already known that ependymal cells express Wnt 8b during development and in

the adult animal brain (Shimogori et al., 2004). Also, our results shows, that ependymal

cells accumulates Wnt 8b in the basal part of the cilia.

The morphology of the ependymal cells during development indicates that they

could have secretory functions. Floor plate ependymal cells in the neural tube possess

vesicles and multivesicular bodies (Yoshioka and Tanaka, 1989). Secretory droplets are

present at the apical surface of rat third ventricles ependyma prior acquisitions of cilia

(Booz, 1975). In the adult rabbit, aqueductal ependymal cells possess surface blebs and

vesicles of various sizes (Meller and Denis, 1993). Ependymal cells in adult humans show

immunoreactivity for granulophysin, a member protein associated with exocytosis

(Hatskelzon et al., 1993) and rat ependymal cells expresses mRNA for the secretory

proteins chromogranin B and secretogranin (Gee at al., 1993). Also possibly involved in

secretion is sulfated glycoprotein – 2 (clusterin) which is expressed at high levels in rat

ependymal (Danik et al., 1993; Senut et al., 1992).

The hypothesis, that ependymal cells secrete Wnt 8b, is consistent with a large body

of evidence indicating that mature ependymal cells express and synthesize Wnt 8b, also

possess proteins and organelle structure required for secretion.

52

Fig. 14. A - confocal image of

cultured ependymal cells from the

side, confirms wnt 8b accumulation in

the basal part of the cilia (green -

acetylated tubulin; red – Wnt 8b; scale

bar 2 µm). B - Maturation of Wnt.

Wnts enter into ER, where initial

glycosylation and palmitoylation

occur, and then are transported into the

Golgi apparatus, where further

glycosylation processes occur and are

finally secreted from the cell surface as

a mature and active

form. After secretion,

Wnts are associated

with proteoglycans or

other extracellular

matrix components,

and transported to the

target cells.

.

53

3.3.2 The role of Dishevelled as Wnt signal transducer in the cilia

The Wnts are a family of secreted factors that bind Frizzled receptors to activate

distinct signaling cascades depending on the specific Wnt activator, the receptor and also

the activity of Disheveled (Dvl), a molecular switch between signaling cascades. Upon

activation of the pathway, the subcellular localization of Dvl appears to determine the

final signaling outcome whereby nuclear localization of Dvl, which is required for

canonical Wnt signaling, while membrane bound Dvl favors the PCP pathway (Veeman et

al., 2003; Gerdes et al., 2008).

Fig. 15. Cilia Wnt signaling. Representation of a cilium/basal body, denoting their role in modulating

the balance between canonical and non-canonical PCP signaling. In conditions of normal ciliary/basal body

54

signaling, molecules such as Inversin (Inv) are required to favor PCP over canonical signaling by inhibiting

Dvl and activating the b-catenin destruction complex composed of Axin, GSK3-b and APC. If Dvl-1 (which

is accumulated in the cilia) is not inhibited, it will transmit further canonical Wnt signal. In contrast, if Dvl-

1 is inhibited, membranous DVL-3 should transmit non-canonical Wnt signaling. Perturbation of the basal

body (i.e., mutations in the BBS proteins) or cilia-mediated signal transduction results in decreased PCP and

the concomitant up-regulation of canonical signaling. (Cardenas et al., 2009)

Having this in mind and based on our results, we can suggest that Dvl-1 is likely

acting as canonical Wnt signal transducer; Dvl-3 – nocanonical. We observed intensive

immunoreactivity of Dvl-1 in the nucleus and in the cilia (also weak cytoplasm staining)

in the ependymal cells. Dvl-1 localization in those place shows that it is significant for

canonical Wnt signaling in this type of cells. Dvl-3 showed weak cytoplasm staining and

good signal next to the membrane. These results are not conclusive, but we can suggest

that Dvl-3 is closely relate to the membrane and possesses importance to non-canonical

Wnt signaling in the ependymal cells.

Although, studies making use of gene interruption to knock-out the expression of

Dvl1, Dvl2, or both (Hamblet et al., 2002) in mice (Lijam et al., 1997; Wang et al., 2006)

reveal some level of redundancy, also we think this is apparent in the current study.

Little is known about the regulation of Dvl expression at the levels of translation and

protein stability, but certainly interesting differences must exist among how these three

highly-homologous mammalian Dvls are processed to perform such different functions.

3.3.3 Dishevelled nuclear shuttling

Dvl has been shown to shuttle between the cytoplasm and the nuclear (Fanto et al.,

2000; Habas et al., 2005; Weitzman 2005). A nuclear export sequence (NES) and an

atypical nuclear localization sequence (NLS) were identified right after the DEP domain

and the PDZ domain (Itoh et al., 2005). Disruption of the nuclear localization of Dvl by

NLS mutation specifically impairs the -catenin pathway, but not the non-canonical

pathway. The importance in the canonical pathway of nuclear localization of mammalian

Dvl was also demonstrated (Itoh et al., 2005). In the nucleus, Dvl has been found to

55

interact with c-Jun and -catenin, followed by formation of the stable -catenin/TCF

complex and transcriptional activation of Wnt target genes (Gan et al., 2008).

We showed that portion of Dvl-1 is localized in the cilia (discussed above) and other

portion in the nucleus. Presence of Dvl 1 in the nucleus indicates, that it is “ working” as a

transducer in the canonical Wnt signaling. Dvl-3 and Dvl-2 demonstrated weak nucleus

staining, this indicates that Dvl functions are overlapping at some level.

Therefore, it is likely that there are two pools of Dvl: one translocates to the nucleus

to mediate the canonical signaling, and the other stays in the cytoplasm or moves to the

plasma membrane to mediate both canonical and non-canonical signaling, upon Wnt

stimulation.

Figure 16. Results of immunohistochemical experiments with cultured ependymal cells. Dvl-1

mainly is accumulated in the nucleus of cultured ependymal cells, white arrows indicate nucleus of the cell.

(In red – Dvl-1; scale bar 20 µm)

56

4. Conclusions

Investigation of ependymal cells in vivo and in vitro revealed significant

result to Wnt signaling and potential pharmaceutical targets in ependymal cells. I have

demonstrated that Wnt lingands and signal transducers are localized in the ependymal

cells.

1. Wnt 8b immunoreactivity, in the basal part of the cilia of ependymal cells, is the

most important finding, this is brand new location. It is know, that this part of the

cilia possesses secretion features that is why we believe that WNT 8b may be

secreted from ependymal cells to the cerebro spinal fluid. This could play

important role in prenatal cortical development and repair after injury. These

results are consistent in vivo and in vitro. To conclude, Wnt 8b is very interesting

potential target in ependymal cells.

2. We have demonstrated that Wnt 3a, Wnt 5a, Wnt 7a and Wnt 7b are not

localized in the ependymal cells. Those antibodies did not label cultured

ependymal cells or brain sections (in vivo), but there were some staining in other

brain regions, already reported in the literature. It means these antibodies work.

According to these results, we suggest that these ligands can not be targets for the

drugs, because it might be that there is no expression of these proteins in

ependymal cells. It is advisable to confirm these results with situ hybridization

method.

3. Our study possesses important results for Dishevelled molecules. We reported that

Dvl-1 is accumulated in the nucleus and in the basal part of cilia of ependymal

cells. This localization is not described in the literature. According to Dvl-1

accumulation place we can suggest that it participates in canonical WNT signaling.

It might be that drug mediated canonical Wnt signaling modification can be

achieved through Dvl-1 and it will be important drug target. Dvl-1 may be an

important drug target for treating ependymomas, because in other forms of cancer,

it is known that canonical WNT signaling pathway is over activated. In

ependymomas Dvl-1 may be drug inhibited in order to restore balance of canonical

Wnt signaling.

57

4. Analysis of Dvl-2 antibody staining with fluorescence and confocal microscope

detected no immunoreactivity in the ependymal cells lined all ventricular system

of the brains and in the cells culture. As well, there is no evidence in literature that

this protein is expressed these cells. This might be indicating that Dvl-2 does not

participate in signaling transduction processes during normal conditions in the

ependymal cells. According to this data, we strongly suggest that Dvl-2 can act as

a drug target. It is advisable to confirm these results with situ hybridization

method.

5. Dvl-3 is accumulated on the membrane of the ependymal cells. This localization is

a completely new place. Dvl-3 membrane localization suggests its involvement in

non-canonical WNT signaling, which affects planar cell polarization. According to

these data, Dvl-3 may be a good drug target for treating the diseases, which

display the impaired ciliar activity. An example could be impaired neuronal

migration in parallel with the lateral ventricle. Thus, Dvl-3 may be a good target

for adjusting the degenerative processes, because increased expression of Dvl-3

might accelerate migration of neuroblasts and astrocyte toward damaged areas,

thereby stopping further damage.

58

5. Bibliography:

1. Adachi, K. et al. -catenin signaling promotes proliferation of

progenitor cells in the adult mouse subventricular zone. Stem Cells 25, 2827–2836

(2007).

2. Adam E, Dziegielewska KM, Saunders NR, Schumacher U. (1993);

Neuraminic acid specific lectins as markers of early cortical plate neurons. Int J Dev

Neurosci. 1993 Aug;11(4):451-60.

3. Acarin L, Vela JM, González B, Castellano B (1994); Demonstration of

poly-N-acetyl lactosamine residues in ameboid and ramified microglial cells in rat

brain by tomato lectin binding. J Histochem Cytochem. 1994 Aug;42(8):1033-41.

4. Ahmad-Annuar, A. et al. Signaling across the synapse: a role for Wnt

and Dishevelled in presynaptic assembly and neurotransmitter release. J. Cell Biol.

174, 127–139 (2006).

5. Arai Y, Deguchi K, Takashima S (1998) Vascular endothelial growth

factor in brains with periventricular leukomalacia. Pediatr Neurol 19:45–49

6. Badaut J, Lasbennes F, Magistretti PJ, Regli L.,2002. Aquaporins in

brain: distribution, physiology, and pathophysiology. J Cereb Blood Flow Metab.

2002 Apr;22(4):367-78.

7. Barker N, Clevers H. Mining the Wnt pathway for cancer therapeutics.

Nat Rev Drug Discov. 2006 Dec;5(12):997-1014.

8. Baker, N. E. Localization of transcripts from the wingless gene in

whole Drosophila embryos. Development 103, 289–298 (1988).

9. Bilic, J. et al. Wnt induces LRP6 signalosomes and promotes

dishevelled-dependent LRP6 phosphorylation. Science 316, 1619–1622 (2007).

10. Bystron I, Blakemore C, Rakic P (2008) Development of the human

cerebral cortex: Boulder Committee revisited. Nat Rev Neurosci 9:110–122

11. Brightman MW, Reese TS (1969); Junctions between intimately

apposed cell membranes in the vertebrate brain. J Cell Biol. 1969 Mar;40(3):648-77.

12. Bruni JE. 1998. Ependymal development, proliferation, and functions.

Microsc Res Tech. 1998 Apr 1;41(1):2-13.

59

13. Boisvieux-Ulrich, E., Laine, M.C. & Sandoz, D. The orientation of

ciliary basal bodies in quail oviduct is related to the ciliary beating cycle

commencement. Biol. Cell 55, 147–150 (1985).

14. Boonen, R. A., van Tijn, P. & Zivkovic, D. Wnt signaling in

Alzheimer’ s disease: up or down, that is the question. Ageing Res. Rev. 8, 71–82

(2009).

15. Booz KH. 1975. Secretory phenomena at the ependyma of the IIIrd

ventricle of the embryonic rat. Anat Embryol (Berl). 1975 Aug 9;147(2):143-59.

16. Boutros M, Paricio N, Strutt DI, Mlodzik M. Dishevelled activates JNK

and discriminates between JNK pathways in planar polarity and wingless signaling.

Cell. 1998 Jul 10;94(1):109-18.

17. Boutros M, Mlodzik M. Dishevelled: at the crossroads of divergent

intracellular signaling pathways. Mech Dev. 1999 May;83(1-2):27-37.

18. Bulinski, J.C., J.E. Richards, and G. Piperno. 1988. Posttranslational

modifications of alpha-tubulin: detyrosination and acetylation differentiate

populations of interphase microtubules in cultured-cells. J. Cell Biol. 106:1213–

1220.

19. Brusco A, López-Costa JJ, Tagliaferro P, Pecci Saavedra J.

Serotonergic ependymal fibres in rat and monkey: light and electron microscopic

immunocytochemical study. Biocell. 1998 Aug;22(2):115-22.

20. G. Castelo-Branco, J.Wagner, F.J. Rodriguez, J. Kele, K. Sousa, N.

Rawal, H.A. Pasolli, E. Fuchs, J. Kitajewski, E. Arenas, Differential regulation of

midbrain dopaminergic neuron development by Wnt-1, Wnt-3a, and Wnt-5a, Proc.

Natl. Acad. Sci. USA 100 (2003) 12747–12752.

21. Caruso, A. et al. Inhibition of the canonical Wnt signaling pathway by

apolipoprotein E4 in PC12 cells. J. Neurochem. 98, 364–371 (2006).

22. Capelluto, D.G. et al. The DIX domain targets dishevelled to actin

stress fibres and vesicular membranes. Nature 419, 726–729 (2002).

23. Caricasole, A. et al. The Wnt pathway, cell-cycle activation and -

amyloid: novel therapeutic strategies in Alzheimer’ s disease? Trends Pharmacol.

Sci. 24, 233–238 (2003).

60

24. Cardenas-Rodriguez M, Badano JL. Ciliary biology: understanding the

cellular and genetic basis of human ciliopathies. Am J Med Genet C Semin Med

Genet. 2009 Nov 15;151C(4):263-80.

25. Cerpa, W., Farías, G. G., Fuenzalida, M., Bonansco, C. & Inestrosa, N.

C. Wnt-5a occludes A oligomerinduced depression of glutamatergic transmision in

CA1 pyramidal neurons from hippocampal slices. Mol. Neurodegener. 2010.

26. Cheyette BN, Waxman JS, Miller JR, Takemaru K, Sheldahl LC,

Khlebtsova N, Fox EP, Earnest T, Moon RT. Dapper, a Dishevelled-associated

antagonist of beta-catenin and JNK signaling, is required for notochord formation.

Dev Cell. 2002 Apr;2(4):449-61.

27. Chen D, Zhang J, Li M, Rayburn ER, Wang H, Zhang R. RYBP

stabilizes p53 by modulating MDM2. EMBO Rep. 2009 Feb;10(2):166-72. Epub

2008 Dec 19.

28. Chen, W. et al. Dishevelled 2 recruits b-arrestin 2 to mediate Wnt5A-

stimulated endocytosis of Frizzled 4. Science 301, 1391–1394 (2003).

29. Chenn, A. & Walsh, C. A. Regulation of cerebral cortical size by

control of cell cycle exit in neural precursors. Science 297, 365–369 (2002).

30. Ching W, Nusse R. A dedicated Wnt secretion factor. Cell 2006;

125:432-433

31. Christensen, S. T., Pedersen, L. B., Schneider, L. and Satir, P. (2007).

Sensory cilia and integration of signal transduction in human health and disease.

Traffic 8, 97-109.

32. Cohen, E., Binet, S. & Meininger, V. Ciliogenesis and centriole

formation in the mouse embryonic nervous system. An ultrastructural analysis. Biol.

Cell 62, 165–169 (1988).

33. Classen, A.K., Anderson, K.I., Marois, E. & Eaton, S. Hexagonal

packing of Drosophila wing epithelial cells by the planar cell polarity pathway. Dev.

Cell 9, 805–817 (2005).

34. Colas JF, Schoenwolf GC (2001) Towards a cellular and molecular

understanding of neurulation. Dev Dyn 221:117–145.

35. Cuevas P, Gimenez-Gallego G (2000) Fibroblast growth factor and

hydrocephalus. Neurol Res 22:102–104.

61

36. Dobell, C. (1932). Antony van Leeuwenhoek and his ‘Little Animals’ ,

p. 435. New York: Harcourt, Brace and Co.

37. Dann CE, Hsieh JC, Rattner A, Sharma D, Nathans J, Leahy DJ.

Insights into Wnt binding and signalling from the structures of two Frizzled

cysteine-rich domains. Nature. 2001 Jul 5;412(6842):86-90.

38. Danik M, Chabot JG, Hassan-Gonzalez D, Suh M, Quirion R..

Localization of sulfated glycoprotein-2/clusterin mRNA in the rat brain by in situ

hybridization. J Comp Neurol. 1993 Aug 8;334(2):209-27.

39. Duan, X., Kang, E., Liu, C. Y., Ming, G. L. & Song, H. Development

of neural stem cell in the adult brain. Curr. Opin. Neurobiol. 18, 108–115 (2008).

40. De Ferrari, G. V. & Inestrosa, N. C. Wnt signaling function in

Alzheimer’ s disease. Brain Res. Brain Res. Rev. 33, 1–12 (2000).

41. De Ferrari, G. V. et al. Activation of Wnt signaling rescues

neurodegeneration and behavioral impairments induced by -amyloid fibrils. Mol.

Psychiatry 8, 195–208 (2003).

42. Del Bigio MR., 1995. The ependyma: a protective barrier between

brain and cerebrospinal fluid. Glia. 1995 May;14(1):1-

43. Frautschy SA, Walicke PA, Baird A (1991) Localization of basic

fibroblast growth factor and its mRNA after CNS injury. Brain Res 553:291–299.

44. Fagotto F, Jho E, Zeng L, Kurth T, Joos T, Kaufmann C, Costantini F.

Domains of axin involved in protein-protein interactions, Wnt pathway inhibition,

and intracellular localization. J Cell Biol. 1999 May 17;145(4):741-56.

45. Farias, G. G. et al. M1 muscarinic receptor activation protects neurons

from -amyloid toxicity. A role for Wnt signaling pathway. Neurobiol. Dis. 17, 337–

348 (2004).

46. Fanto M, Weber U, Strutt DI, Mlodzik M. Nuclear signaling by Rac

and Rho GTPases is required in the establishment of epithelial planar polarity in the

Drosophila eye. Curr Biol. 2000 Aug 24;10(16):979-88.

47. Figarella-Branger D, Lepidi H, Poncet C, Gambarelli D, Bianco N,

Rougon G, Pellissier JF. Differential expression of cell adhesion molecules (CAM),

neural CAM and epithelial cadherin in ependymomas and choroid plexus tumors.

Acta Neuropathol. 1995;89(3):248-57.

62

48. Frisch, D. & Farbman, A.I. Development of order during ciliogenesis.

Anat. Rec. 162, 221–232 (1968).

49. Fujii N, You L, Xu Z, Uematsu K, Shan J, He B, Mikami I, Edmondson

LR, Neale G, Zheng J, Guy RK, Jablons DM. An antagonist of dishevelled protein-

protein interaction suppresses beta-catenin-dependent tumor cell growth. Cancer

Res. 2007 Jan 15;67(2):573-9.

50. Fuxe K, Tinner B, Zoli M, Pettersson RF, Baird A, Biagini G, Chadi G,

Agnati LF (1996) Computer-assisted mapping of basic fibroblast growth factor

immunoreactive nerve cell populations in the rat brain. J Chem Neuroanat 11:13–35

51. Galli LM, Barnes TL, Secrest SS, Kadowaki T, Burrus LW. Porcupine-

mediated lipid-modification regulates the activity and distribution of Wnt proteins in

the chick neural tube. Development. 2007 Sep;134(18):3339-48.

52. Gao, Z. et al. Neurod1 is essential for the survival and maturation of

adult-born neurons. Nature Neurosci. 12, 1090–1092 (2009).

53. Gee P, Rhodes CH, Fricker LD, Angeletti RH. Expression of

neuropeptide processing enzymes and neurosecretory proteins in ependyma and

choroid plexus epithelium. Brain Res. 1993 Jul 23;617(2):238-48.

54. Gerdes JM, Katsanis N. 2008. Ciliary function and Wnt signal

modulation. Curr Top Dev Biol 85:175–195.

55. Gerdes, J.M. et al. Disruption of the basal body compromises

proteasomal function and perturbs intracellular Wnt response. Nat. Genet. 39, 1350–

1360 (2007).

56. Geng L, Okuhara D, Yu Z, Tian X, Cai Y, et al. 2006. Polycystin-2

traffics to cilia independently of polycystin-1 by using an N-terminal RVxP motif. J.

Cell Sci. 119:1383–95

57. Gilula NB, Satir P. 1972. The ciliary necklace. A ciliary membrane

specialization. J. Cell Biol. 53:494–509

58. Grandy D, Shan J, Zhang X, Rao S, Akunuru S, Li H, Zhang Y,

Alpatov I, Zhang XA, Lang RA, Shi DL, Zheng JJ. Discovery and characterization

of a small molecule inhibitor of the PDZ domain of dishevelled. J Biol Chem. 2009

Jun 12;284(24):16256-63. Epub 2009 Apr 21.

63

59. Gonzalez AM, Berry M, Maher PA, Logan A, Baird A (1995) A

comprehensive analysis of the distribution of FGF-2 and FGFR1 in the rat brain.

Brain Res 701:201–226.

60. Habas R, Dawid IB, He X: Coactivation of Rac and Rho by

Wnt/Frizzled signaling is required for vertebrate gastrulation. Genes Dev 2003,

17:295-309.

61. Habas R, Dawid IB. Dishevelled and Wnt signaling: is the nucleus the

final frontier? J Biol. 2005;4(1):2. Epub 2005 Feb 17.

62. Habas R, Kato Y, He X: Wnt/Frizzled activation of Rho regulates

vertebrate gastrulation and requires a novel Formin homology protein Daam1. Cell

2001, 107:843-854.

63. Hayamizu TF, Chan PT, Johanson CE (2001) FGF-2 immunoreactivity

in adult rat ependyma and choroid plexus: responses to global forebrain ischemia

and intraventricular FGF-2. Neurol Res 23:353–358

64. Hall, A. C., Lucas, F. R. & Salinas, P. C. Axonal remodeling and

synaptic differentiation in the cerebellum is regulated by WNT-7a signaling. Cell

100, 525–535 (2000).

65. Hamblet NS, Lijam N, Ruiz-Lozano P, Wang J, Yang Y, Luo Z, Mei L,

Chien KR, Sussman DJ, Wynshaw-Boris A. Dishevelled 2 is essential for cardiac

outflow tract development, somite segmentation and neural tube closure.

Development 2002;129:5827–5838. [PubMed: 12421720]

66. Hatskelzon L, Dalal BI, Shalev A, Robertson C, Gerrard JM. 1993.

Wide distribution of granulophysin epitopes in granules of human tissues. Lab

Invest. 1993 May;68(5):509-19.

67. Hausmann G, Bänziger C, Basler K. Helping Wingless take flight: how

WNT proteins are secreted. Nat Rev Mol Cell Biol. 2007 Apr;8(4):331-6. Epub

2007 Mar 7.

68. Hauwel M, Furon E, Canova C, Griffiths M, Neal J, Gasque P (2005)

Innate (inherent) control of brain infection, brain inflammation and brain repair: the

role of microglia, astrocytes, ‘‘protective’ ’ glial stem cells and stromal ependymal

cells. Brain Res Brain Res Rev 48:220–233

64

69. Hebert JM, Fishell G (2008) The genetics of early telencephalon

patterning: some assembly required. Nat Rev Neurosci 9:678–685.

70. He, P. & Shen, Y. Interruption of -catenin signaling reduces

neurogenesis in Alzheimer’ s disease. J. Neurosci. 29, 6545– 6557 (2009).

71. He X, Saint-Jeannet JP, Wang Y, Nathans J, Dawid I, Varmus H. A

member of the Frizzled protein family mediating axis induction by Wnt-5A.

Science. 1997 Mar 14;275(5306):1652-4.

72. He X, Semenov M, Tamai K, Zeng X: LDL receptor-related proteins 5

and 6 in Wnt/beta-catenin signaling: arrows point the way. Development 2004,

131:1663-1677.

73. Herz J, Bock HH. Lipoprotein receptors in the nervous system. Annu

Rev Biochem. 2002;71:405-34. Epub 2001 Nov 9.

74. Hirabayashi, Y. et al. The Wnt/-catenin pathway directs neuronal

differentiation of cortical neural precursor cells. Development 131, 2791–2801

(2004).

75. Hong DH, Yue G, Adamian M, Li T. 2001. Retinitis pigmentosa

GTPase regulator (RPGRr)-interacting protein is stably associated with the

photoreceptor ciliary axoneme and anchors RPGR to the connecting cilium. J. Biol.

Chem. 276:12091–99.

76. Hong DH, Pawlyk B, Sokolov M, Strissel KJ, Yang J, et al. 2003.

RPGR isoforms in photoreceptor connecting cilia and the transitional zone of motile

cilia. Invest. Ophthalmol.Vis. Sci. 44:2413–21.

77. Horton BN, Solanki RB, Kulesza P, Ardelt AA (2009) Localization of

angiopoietin-1 and Tie2 immunoreactivity in rodent ependyma and adjacent blood

vessels suggests functional relationships. J Histochem Cytochem.

doi:10.1369/jhc.2009.954610.

78. Iwata T, Hevner RF (2009) Fibroblast growth factor signaling in

development of the cerebral cortex. Dev Growth Differ 51:299–323

79. Yang-Snyder J, Miller JR, Brown JD, Lai CJ, Moon RT. A frizzled

homolog functions in a vertebrate Wnt signaling pathway. Curr Biol. 1996 Oct

1;6(10):1302-6.

65

80. Inestrosa, N. C. & Toledo, E. M. The role of Wnt signaling in neuronal

dysfunction in Alzheimer’ s disease. Mol. Neurodegener. 3, 9 (2008).

81. Yamadori T, Nara K. (1979); The directions of ciliary beat on the wall

of the lateral ventricle and the currents of the cerebrospinal fluid in the brain

ventricles. Scan Electron Microsc. 1979;(3):335-40.

82. Yamamoto T, Kardami E, Nagy JI., 1995. Basic fibroblast growth

factor in rat brain: localization to glial gap junctions correlates with connexin43

distribution. Brain Res. 1991 Jul 19;554(1-2):336-43.

83. Yanagawa S, van Leeuwen F, Wodarz A, Klingensmith J, Nusse. The

dishevelled protein is modified by wingless signaling in Drosophila. Genes Dev.

1995 May 1;9(9):1087-97.

84. Ye X, Carp RI (2002) Increase of acidic fibroblast growth factor in the

brains of hamsters infected with either 263K or 139H strains of scrapie. J Mol

Neurosci 18:179–188.

85. Yoshioka T, Tanaka O. 1989. Ultrastructural and cytochemical

characterisation of the floor plate ependyma of the developing rat spinal cord. J

Anat. 1989 Aug;165:87-100.

86. Yu, A. et al. Association of Dishevelled with the clathrin AP-2 adaptor

is required for Frizzled endocytosis and planar cell polarity signaling. Dev. Cell 12,

129–141 (2007).

87. Yu S, Tooyama I, Ding WG, Kitasato H, Kimura H (1995)

Immunohistochemical localization of glucose transporters (GLUT1 and GLUT3) in

the rat hypothalamus. Obes Res 3(Suppl 5):753S–776S.

88. Yu, J. M., Kim, J. H., Song, G. S. & Jung, J. S. Increase in proliferation

and differentiation of neural progenitor cells isolated from postnatal and adult mice

brain by Wnt-3a and Wnt-5a. Mol. Cell Biochem. 288, 17–28 (2006).

89. Jekely, G. and Arendt, D. (2006). Evolution of intraflagellar transport

from coated vesicles and autogenous origin of the eukaryotic cilium. BioEssays 28,

191-198.

90. Jessberger, S. et al. Dentate gyrus-specific knockdown of adult

neurogenesis impairs spatial and object recognition memory in adult rats. Learn.

Mem. 16, 147–154 (2009).

66

91. Johnson, N. T., Villalon, M., Royce, F. H., Hard, R. and Verdugo, P.

(1991). Autoregulation of beat frequency in respiratory ciliated cells. Demonstration

by viscous loading. Am. Rev. Respir. Dis. 144, 1091-1094.

92. Keiji Itoh, Barbara K Brott, Gyu-Un Bae, Marianne J Ratcliffe, and

Sergei Y Sokol. Nuclear localization is required for Dishevelled function in Wnt/-

catenin signaling. J Biol. 2005; 4(1): 3. Published online 2005 February 15. doi:

10.1186/jbiol20.

93. Keller R: Shaping the vertebrate body plan by polarized embryonic cell

movements. Science 2002, 298:1950-1954.

94. Kennerdell JR, Carthew RW. Use of dsRNA-mediated genetic

interference to demonstrate that frizzled and frizzled 2 act in the wingless pathway.

Cell. 1998 Dec 23;95(7):1017-26.

95. Kim, J.C. et al. The Bardet-Biedl protein BBS4 targets cargo to the

pericentriolar region and is required for microtubule anchoring and cell cycle

progression. Nat. Genet. 36, 462–470 (2004).

96. Kimura J, Suda Y, Kurokawa D, Hossain ZM, Nakamura M, Takahashi

M, Hara A, Aizawa S. 2005. Emx2 and Pax6 function in cooperation with Otx2 and

Otx1 to develop caudal forebrain primordium that includes future archipallium. J

Neurosci 25:5097–5108.

97. Kishida, S. et al. Dvl regulates endo- and exocytotic processes through

binding to synaptotagmin. Genes Cells 12, 49–61 (2007).

98. Kobayashi M, Nikami H, Morimatsu M, Saito M (1996) Expression

and localization of insulin-regulatable glucose transporter (GLUT4) in rat brain.

Neurosci Lett 213:103–106.

99. Komekado H, Yamamoto H, Chiba T, Kikuchi Glycosylation and

palmitoylation of Wnt-3a are coupled to produce an active form of Wnt-3a. A.

Genes Cells. 2007 Apr;12(4):521-34.

100. Korkut C, Budnik V. WNTs tune up the neuromuscular junction. Nat

Rev Neurosci. 2009 Sep;10(9):627-34.

101. Kowalevsky, A. (1867). Entwickelungsgeschichte des Amphioxus

lanceolatus. Memoires de l’ Academie Imperiale des Sciences de St.-Petersbourg

VII. 11, 1-17.

67

102. Kramer-Zucker, A.G. et al. Cilia-driven fluid flow in the zebrafish

pronephros, brain and Kupffer’ s vesicle is required for normal organogenesis.

Development 132, 1907–1921 (2005).

103. Kuhl M: Non-canonical Wnt signaling in Xenopus: regulation of axis

formation and gastrulation. Semin Cell Dev Biol 2002, 13:243-249.

104. Kuwabara, T. et al. Wnt-mediated activation of NeuroD1 and retro-

elements during adult neurogenesis. Nature Neurosci. 12, 1097–1105 (2009).

105. Lacor, P. N. et al. A oligomer-induced aberrations in synapse

composition, shape, and density provide a molecular basis for loss of connectivity in

Alzheimer’ s disease. J. Neurosci. 27, 796–807 (2007).

106. Lako MS, Lindsay S, Bullen P, Wilson DI, Robson SC, Strachan T.

1998. A novel mammalian wnt gene, WNT8B, shows brain-restricted expression in

early development, with sharply delimited expression boundaries in the developing

forebrain. Hum Mol Genet 7:813–822.

107. Langerhans, P. (1876). Zur Anatomie des Amphioxus. Archiv fur

Mikrokopische Anatomie 12, 290-348.

108. Lange BM, Faragher AJ, March P, Gull K (2000) Centriole duplication

and maturation in animal cells. Curr Top Dev Biol 49:235–249.

109. Lanzetti, L. Actin in membrane trafficking. Curr. Opin. Cell Biol. 19,

453–458 (2007)

110. Lee HJ, Wang NX, Shao Y, Zheng JJ. Identification of tripeptides

recognized by the PDZ domain of Dishevelled. Bioorg Med Chem. 2009 Feb

15;17(4):1701-8. Epub 2009 Jan 3.

111. Li X, Kong H, Wu W, Xiao M, Sun X, Hu G. Aquaporin-4 maintains

ependymal integrity in adult mice. Neuroscience. 2009 Aug 4;162(1):67-77. Epub

2009 Apr 22.

112. Lie, D. C., Song, H., Colamarino, S. A., Ming, G. L. & Gage, F. H.

Neurogenesis in the adult brain: new strategies for central nervous system diseases.

Annu. Rev. Pharmacol. Toxicol. 44, 399–421 (2004).

113. Li AJ,OomuraY,SasakiK, SuzukiK,Tooyama I,HanaiK,Kimura H, Hori

T (1998) A single pre-training glucose injection induces memory facilitation in

68

rodents performing various tasks: contribution of acidic fibroblast growth factor.

Neuroscience 85:785–794

114. Lijam N, Paylor R, McDonald MP, Crawley JN, Deng CX, Herrup K,

Stevens KE, Maccaferri G, McBain CJ, Sussman DJ, Wynshaw-Boris A. Social

interaction and sensorimotor gating abnormalities in mice lacking Dvl1. Cell

1997;90:895–905. [PubMed: 9298901]

115. Ling EA, Kaur C, Lu J (1998) Origin, nature, and some functional

considerations of intraventricular macrophages, with special reference to the

epiplexus cells. Microsc Res Tech 41:43–56.

116. Liu C, Kato Y, Zhang Z, Do VM, Yankner BA, He X: beta-Trcp

couples beta-catenin phosphorylation-degradation and regulates Xenopus axis

formation. Proc Natl Acad Sci USA 1999, 96:6273-6278.

117. Liu G, Bafico A, Harris VK, Aaronson SA. A novel mechanism for

Wnt activation of canonical signaling through the LRP6 receptor. Mol Cell Biol.

2003 Aug;23(16):5825-35.

118. Liu T, DeCostanzo AJ, Liu X, Wang Hy, Hallagan S, Moon RT,

Malbon CC. G protein signaling from activated rat frizzled-1 to the beta-catenin-

Lef-Tcf pathway. Science. 2001 Jun 1;292(5522):1718-22.

119. Logan CY, Nusse R: The Wnt signaling pathway in development and

disease. Annu Rev Cell Dev Biol 2004, 20:781-810.

120. Lorenzo, I. M., Liedtke, W., Sanderson, M. J. and Valverde, M. A.

(2008). TRPV4 channel participates in receptor-operated calcium entry and ciliary

beat frequency regulation in mouse airway epithelial cells. Proc. Natl. Acad. Sci.

USA 105, 12611-12616.

121. Lucas, F.R., and P.C. Salinas. 1997. WNT-7a induces axonal

remodeling and increases synapsin I levels in cerebellar neurons. Dev. Biol. 192:31–

44.

122. Maharaj AS, Walshe TE, Saint-Geniez M, Venkatesha S, Maldonado

AE, Himes NC, Matharu KS, Karumanchi SA, D’ Amore PA (2008) VEGF and

TGF-beta are required for the maintenance of the choroid plexus and ependyma. J

Exp Med 205:491–501.

69

123. Magdesian, M. H. et al. Amyloid- binds to the extracellular cysteine-

rich domain of Frizzled and inhibits Wnt/-catenin signaling. J. Biol. Chem. 283,

9359–9368 (2008).

124. Marlow F, Topczewski J, Sepich D, Solnica-Krezel L: Zebrafish Rho

kinase 2 acts downstream of Wnt11 to mediate cell polarity and effective

convergence and extension movements. Curr Biol 2002, 12:876-884.

125. Mao J, Wang J, Liu B, Pan W, Farr GH 3rd, Flynn C, Yuan H, Takada

S, Kimelman D, Li L, Wu D. Low-density lipoprotein receptor-related protein-5

binds to Axin and regulates the canonical Wnt signaling pathway. Mol Cell. 2001

Apr;7(4):801-9.

126. McMahon, A. P. & Moon, R. T. int-1 — a proto-oncogene involved in

cell signalling. Development 107 (Suppl.), 161–167 (1989).

127. Meller ST, Dennis BJ. 1993. A scanning and transmission electron

microscopic analysis of the cerebral aqueduct in the rabbit. Anat Rec. 1993

Sep;237(1):124-40.

128. Michaloudi HC, Papadopoulos GC (1996) Catecholaminergic and

serotoninergic fibres innervate the ventricular system of the hedgehog CNS. J Anat

189(Pt 2):273–283.

129. Ming, G. L. & Song, H. Adult neurogenesis in the mammalian central

nervous system. Annu. Rev. Neurosci. 28, 223–250 (2005).

130. Mitchell, B., Jacobs, R., Li, J., Chien, S. & Kintner, C. A positive

feedback mechanism governs the polarity and motion of motile cilia. Nature 447,

97–101 (2007).

131. Miller JR, Hocking AM, Brown JD, Moon RT: Mechanism and

function of signal transduction by the Wnt/_-catenin and Wnt/Ca2+ pathways.

Oncogene 1999, 18:7860-7872.

132. Mirzadeh Z, Merkle FT, Soriano-Navarro M, Garcia-Verdugo JM,

Alvarez-Buylla A., 2008. Neural stem cells confer unique pinwheel architecture to

the ventricular surface in neurogenic regions of the adult brain. Cell Stem Cell. 2008

Sep 11;3(3):265-78

133. Mobasheri A, Wray S, Marples D. Distribution of AQP2 and AQP3

water channels in human tissue microarrays. J Mol Histol. 2005 Feb;36(1-2):1-14.

70

134. Mobasheri A, Marples D. Expression of the AQP-1 water channel in

normal human tissues: a semiquantitative study using tissue microarray technology.

Am J Physiol Cell Physiol. 2004 Mar;286(3):C529-37. Epub 2003 Oct 30.

135. Mudher, A. & Lovestone, S. Alzheimer’ s disease-do tauists and baptists

finally shake hands? Trends Neurosci. 25, 22–26 (2002).

136. Muller, O. F. (1786). Animalcula infusoria; fluvia tilia et marina, que

detexit, systematice descripsit et ad vivum delineari curavit. Havniae: Typis N.

Molleri.

137. Murata K, Mitsuoka K, Hirai T, Walz T, Agre P, Heymann JB, Engel

A, Fujiyoshi Y. 2000. Structural determinants of water permeation through

aquaporin-1. Nature. 2000 Oct 5;407(6804):599-605.

138. Nachury, M.V. et al. A core complex of BBS proteins cooperates with

the GTPase Rab8 to promote ciliary membrane biogenesis. Cell 129, 1201–1213

(2007).

139. Nusse, R. Wnt signaling and stem cell control. Cell Res. 18, 523–527

(2008).

140. Nunes, P. V., Forlenza, O. V. & Gattaz, W. F. Lithium and risk for

Alzheimer’ s disease in elderly patients with bipolar disorder. Br. J. Psychiatry 190,

359–360 (2007).

141. Nusse, R. & Varmus, H. E. Many tumors induced by the mouse

mammary tumor virus contain a provirus integrated in the same region of the host

genome. Cell 31, 99–109 (1982).

142. Nguyen T, Chin WC, O’ Brien JA, Verdugo P, Berger AJ (2001)

Intracellular pathways regulating ciliary beating of rat brain ependymal cells. J

Physiol 531:131–140.

143. Nourhaghighi N, Teichert-Kuliszewska K,Davis J, Stewart DJ,Nag S

(2003) Altered expression of angiopoietins during blood-brain barrier breakdown

and angiogenesis. Lab Invest 83:1211–1222.

144. Oishi, I., Kawakami, Y., Raya, A., Callol-Massot, C. & Izpisua

Belmonte, J.C. Regulation of primary cilia formation and left-right patterning in

zebrafish by a noncanonical Wnt signaling mediator, duboraya. Nat. Genet. 38,

1316–1322 (2006).

71

145. Oomura Y, Sasaki K, Suzuki K, Muto T, Li AJ, Ogita Z, Hanai K,

Tooyama I, Kimura H, Yanaihara N (1992) A new brain glucosensor and its

physiological significance. Am J Clin Nutr 55:278S–282S

146. Pinson KI, Brennan J, Monkley S, Avery BJ, Skarnes WC. An LDL-

receptor-related protein mediates Wnt signalling in mice. Nature. 2000 Sep

28;407(6803):535-8.

147. Panizzi, J.R., Jessen, J.R., Drummond, I.A. & Solnica-Krezel, L. New

functions for a vertebrate Rho guanine nucleotide exchange factor in ciliated

epithelia. Development 134, 921–931 (2007).

148. Patapoutian, A., and L.F. Reichardt. 2000. Roles of Wnt proteins in

neural development and maintenance. Curr. Opin. Neurobiol. 10:392–399.

149. Park, S. C., Yibchok-Anun, S., Cheng, H., Young, T. F., Thacker, E. L.,

Minion, F. C., Ross, R. F. and Hsu, W. H. (2002). Mycoplasma hyopneumoniae

increases intracellular calcium release in porcine ciliated tracheal cells. Infect.

Immun. 70, 2502-2506.

150. Park TJ, Mitchell BJ, Abitua PB, Kintner C, Wallingford JB.

Dishevelled controls apical docking and planar polarization of basal bodies in

ciliated epithelial cells. Nat Genet. 2008 Jul;40(7):871-9. Epub 2008 Jun 15.

151. Park, T.J., Gray, R.S., Sato, A., Habas, R. & Wallingford, J.B.

Subcellular localization and signaling properties of dishevelled in developing

vertebrate embryos. Curr. Biol. 15, 1039–1044 (2005).

152. Parr BA, Shea MJ, Vassileva G, McMahon AP. 1993. Mouse Wnt

genes exhibit discrete domains of expression in the early embryonic CNS and limb

buds. Development. 1993 Sep;119(1):247-61.

153. Paulson JC. (1989); Glycoproteins: what are the sugar chains for?;

Trends Biochem Sci. 1989 Jul;14(7):272-6.

154. Rodriguez-Perez LM, Perez-Martin M, Jimenez AJ, Fernandez- Llebrez

P (2003) Immunocytochemical characterisation of the wall of the bovine lateral

ventricle. Cell Tissue Res 314:325–335

155. Prochnow N, Dermietzel R., 2008. Connexons and cell adhesion: a

romantic phase. Histochem Cell Biol. 2008 Jul;130(1):71-7. Epub 2008 May 15.

72

156. Prothmann C, Wellard J, Berger J, Hamprecht B, Verleysdonk S (2001)

Primary cultures as a model for studying ependymal functions: glycogen metabolism

in ependymal cells. Brain Res 920:74–83.

157. Redies C, Takeichi M. (1996); Cadherins in the developing central

nervous system: an adhesive code for segmental and functional subdivisions. Dev

Biol. 1996 Dec 15;180(2):413-23

158. Roth Y, Kimhi Y, Edery H, Aharonson E, Priel Z.(1985); Ciliary

motility in brain ventricular system and trachea of hamsters. Brain Res. 1985 Mar

25;330(2):291-7.

159. Ross, A.J. et al. Disruption of Bardet-Biedl syndrome ciliary proteins

perturbs planar cell polarity in vertebrates. Nat. Genet. 37, 1135–1140 (2005).

160. Rulifson EJ, Wu CH, Nusse R. Pathway specificity by the bifunctional

receptor frizzled is determined by affinity for wingless. Mol Cell. 2000 Jul;6(1):117-

26. Genes Dev. 1996 Sep 1;10(17):2189-97.

161. Sanderson, M. J. and Dirksen, E. R. (1986). Mechanosensitivity of

cultured ciliated cells from the mammalian respiratory tract: implications for the

regulation of mucociliary transport. Proc. Natl. Acad. Sci. USA 83, 7302-7306.

162. Salathe, M. (2006). Regulation of mammalian ciliary beating. Annu.

Rev. Physiol. 69, 401-422.

163. Sarnat HB (1998) Histochemistry and immunocytochemistry of the

developing ependyma and choroid plexus. Microsc Res Tech 41:14–28.

164. Sarnat HB (1992) Regional differentiation of the human fetal

ependyma: immunocytochemical markers. J Neuropathol Exp Neurol 51:58–75.

165. Sarnat HB (1992) Role of human fetal ependyma. Pediatr Neurol

8:163–178

166. Sarnat HB (1995) Ependymal reactions to injury. A review. J

Neuropathol Exp Neurol 54:1–15.

167. Satir P, Guerra C. 2003. Control of ciliary motility: a unifying

hypothesis. Europ. J. Protistol. 39:410–15

168. Satir, P., Mitchell, D. R. and Jékely, G. (2008). How did the cilium

evolve? Curr. Top. Dev. Biol. 85, 63-82.

73

169. Sawa H, Lobel L, Horvitz HR. The Caenorhabditis elegans gene lin-17,

which is required for certain asymmetric cell divisions, encodes a putative seven-

transmembrane protein similar to the Drosophila frizzled protein.

170. Schwarz-Romond, T., Merrifield, C., Nichols, B.J. & Bienz, M. The

Wnt signaling effector Dishevelled forms dynamic protein assemblies rather than

stable associations with cytoplasmic vesicles. J. Cell Sci. 118, 5269–5277 (2005).

171. Schwarz-Romond T, Fiedler M, Shibata N, Butler PJ, Kikuchi A,

Higuchi Y, Bienz M. The DIX domain of Dishevelled confers Wnt signaling by

dynamic polymerization. Nat Struct Mol Biol. 2007 Jun;14(6):484-92. Epub 2007

May 27.

172. Shankar, G. M. et al. Amyloid-�

protein dimers isolated directly from

Alzheimer’ s brains impair synaptic plasticity and memory. Nature Med. 14, 837–

842 (2008).

173. Schwarz-Romond T, Metcalfe C, Bienz M Dynamic recruitment of axin

by Dishevelled protein assemblies. J Cell Sci. 2007 Jul 15;120(Pt 14):2402-12.

174. Senut MC, Jazat F, Choi NH, Lamour Y. Protein SP40,40-like

Immunoreactivity in the Rat Brain: Progressive Increase With Age. Eur J Neurosci.

1992;4(10):917-928.

175. Shimogori T, VanSant J, Paik E, Grove EA. 2004. Members of the

Wnt, Fz, and Frp gene families expressed in postnatal mouse cerebral cortex. J

Comp Neurol. 2004 Jun 7;473(4):496-510.

176. Shah, A. S., Ben-Shahar, Y., Moninger, T. O., Kline, J. N. and Welsh,

M. J. (2009). Motile cilia of human airway epithelia are chemosensory. Science 325,

1131-1134.

177. Shan J, Shi DL, Wang J, Zheng J. Identification of a specific inhibitor

of the dishevelled PDZ domain. Biochemistry. 2005 Nov 29;44(47):15495-503.

178. Sheldahl LC, Slusarski DC, Pandur P, Miller JR, Kühl M, Moon RT.

Dishevelled activates Ca2+ flux, PKC, and CamKII in vertebrate embryos. J Cell

Biol. 2003 May 26;161(4):769-77.

179. Semënov MV, Tamai K, Brott BK, Kühl M, Sokol S, He X. Head

inducer Dickkopf-1 is a ligand for Wnt coreceptor LRP6. Curr Biol. 2001 Jun

26;11(12):951-61.

74

180. Simons, M. et al. Inversin, the gene product mutated in

nephronophthisis type II, functions as a molecular switch between Wnt signaling

pathways. Nat. Genet. 37, 537–543 (2005).

181. Silva-Alvarez C, Carrasco M, Balmaceda-Aguilera C, Pastor P, Garcia

Mde L, Reinicke K, Aguayo L, Molina B, Cifuentes M, Medina R, Nualart F (2005)

Ependymal cell differentiation and GLUT1 expression is a synchronous process in

the ventricular wall. Neurochem Res 30:1227–1236.

182. Small, S. A. & Duff, K. Linking A and tau in late-onset Alzheimer’ s

disease: a dual pathway hypothesis. Neuron 60, 534–542 (2008).

183. Smalley MJ, Signoret N, Robertson D, Tilley A, Hann A, Ewan K,

Ding Y, Paterson H, Dale TC. Dishevelled (Dvl-2) activates canonical Wnt

signalling in the absence of cytoplasmic puncta. J Cell Sci. 2005 Nov 15;118(Pt

22):5279-89. Epub 2005 Nov 1.

184. Spassky N, Merkle FT, Flames N, Tramontin AD, García-Verdugo JM,

Alvarez-Buylla A., 2005, Adult ependymal cells are postmitotic and are derived

from radial glial cells during embryogenesis. J Neurosci. 2005 Jan 5;25(1):10-8.

185. Steiner J, Bernstein HG, Bielau H, Berndt A, Brisch R, Mawrin C,

Keilhoff G, Bogerts B (2007) Evidence for a wide extraastrocytic distribution of

S100B in human brain. BMC Neurosci 8:2

186. Sorokin, S.P. Reconstructions of centriole formation and ciliogenesis in

mammalian lungs. J. Cell Sci. 3, 207–230 (1968).

187. Sussman, D.J., J. Klingensmith, P. Salinas, P.S. Adams, R. Nusse, and

N. Perrimon. 1994. Isolation and characterization of a mouse homolog of the

Drosophila segment polarity gene dishevelled. Dev. Biol. 166:73–86.

188. Tamai K, Zeng X, Liu C, Zhang X, Harada Y, Chang Z, He X. A

mechanism for Wnt coreceptor activation. Mol Cell. 2004 Jan 16;13(1):149-56.

189. Takada R, Satomi Y, Kurata T, Ueno N, Norioka S, Kondoh H, Takao

T, Takada S. Monounsaturated fatty acid modification of Wnt protein: its role in

Wnt secretion. Dev Cell. 2006 Dec;11(6):791-801.

190. Theil T, Aydin S, Koch S, Grotewold L, Ru¨ ther U. 2002. Wnt and

Bmp signaling cooperatively regulate graded Emx2 expression in the dorsal

telencephalon. Development 129:3045–3054.

75

191. Tonchev AB, Yamashima T, Guo J, Chaldakov GN, Takakura N (2007)

Expression of angiogenic and neurotrophic factors in the progenitor cell niche of

adult monkey subventricular zone. Neuroscience 144:1425–1435

192. Tole S, Ragsdale CW, Grove EA. 2000. Dorsoventral patterning of the

telencephalon is disrupted in the mouse mutant extra-toes (J). Dev Biol 217:254–

265.

193. Toledo, E. M. & Inestrosa, N. C. Activation of Wnt signaling by

lithium and rosiglitazone reduced spatial memory impairment and

neurodegeneration in brains of APPswe/PSEN1�E9 mouse model of Alzheimer’ s

disease. Mol. Psychiatry 21 Jul 2009 (doi:10.1038/ mp.2009.72).

194. Tuomanen E. 1990. The surface of mammalian respiratory cilia. In

Ciliary and Flagellar Membranes, ed. RA Bloodgood, pp. 363–88. New York:

Plenum.

195. Umbhauer M, Djiane A, Goisset C, Penzo-Méndez A, Riou JF, Boucaut

JC, Shi DL. The C-terminal cytoplasmic Lys-thr-X-X-X-Trp motif in frizzled

receptors mediates Wnt/beta-catenin signalling. EMBO J. 2000 Sep 15;19(18):4944-

54.

196. Veeman MT, Axelrod JD, Moon RT: A second canon. Functions and

mechanisms of beta-catenin-independent Wnt signaling. Dev Cell 2003, 5:367-377.

197. Verleysdonk S, Kistner S, Pfeiffer-Guglielmi B, Wellard J, Lupescu A,

Laske J, Lang F, Rapp M, Hamprecht B (2005) Glycogen metabolism in rat

ependymal primary cultures: regulation by serotonin. Brain Res 1060:89–99.

198. Vigh B, Manzano e Silva MJ, Frank CL, Vincze C, Czirok SJ, Szabo A,

Lukats A, Szel A (2004) The system of cerebrospinal fluid-contacting neurons. Its

supposed role in the nonsynaptic signal transmission of the brain. Histol Histopathol

19:607–628.

199. Vives V, Alonso G, Solal AC, Joubert D, Legraverend C (2003)

Visualization of S100B-positive neurons and glia in the central nervous system of

EGFP transgenic mice. J Comp Neurol 457:404–419

200. Wang J, Hamblet NS, Mark S, Dickinson ME, Brinkman BC, Segil N,

Fraser SE, Chen P, Wallingford JB, Wynshaw-Boris A. Dishevelled genes mediate a

76

conserved mammalian PCP pathway to regulate convergent extension during

neurulation. Development 2006;133:1767 1778. [PubMed: 16571627].

201. Wang LX, Yin RX, Sun JB (2008) Effect of Tongxinluo on nestin and

vascular endothehal growth factor mRNA expression in rat brain tissue after cerebral

ischemia-reperfusion injury. Nan Fang Yi Ke Da Xue Xue Bao 28:2131–2135.

202. Wallingford JB, Fraser SE, Harland RM: Convergent extension: the

molecular control of polarized cell movement during embryonic development. Dev

Cell 2002, 2:695-706.

203. Wallingford JB (2006) Planar cell polarity, ciliogenesis and neural tube

defects. Hum Mol Genet 15 Spec No 2:R227–R234.

204. Wallingford, J.B. & Habas, R. The developmental biology of

Dishevelled: an enigmatic protein governing cell fate and cell polarity. Development

132, 4421–4436 (2005).

205. Wexler, E. M., Paucer, A., Kornblum, H. I., Plamer, T. D. &

Geschwind, D. H. Endogenous Wnt signaling maintains neural progenitor cell

potency. Stem Cells 27, 1130–1141 (2009).

206. Wehrli M, Dougan ST, Caldwell K, O'Keefe L, Schwartz S, Vaizel-

Ohayon D, Schejter E, Tomlinson A, DiNardo S. Arrow encodes an LDL-receptor-

related protein essential for Wingless signalling. Nature. 2000 Sep

28;407(6803):527-30. Wharton KA Jr. Runnin' with the Dvl: proteins that associate

with Dsh/Dvl and their significance to Wnt signal transduction. Dev Biol. 2003 Jan

1;253(1):1-17.

207. Weitzman JB. Dishevelled nuclear shuttling. J Biol. 2005;4(1):1. Epub

2005 Feb 16.

208. Wharton KA Jr: Runnin’ with the Dvl: proteins that associate with

Dsh/Dvl and their significance to Wnt signal transduction. Dev Biol 2003, 253:1-17.

209. Willert K, Brown JD, Danenberg E, Duncan AW, Weissman IL, Reya

T, Yates JR 3rd, Nusse R. Wnt proteins are lipid-modified and can act as stem cell

growth factors. Nature. 2003 May 22;423(6938):448-52. Epub 2003 Apr 27.

210. Willert K, Brink M, Wodarz A, Varmus H, Nusse R. Casein kinase 2

associates with and phosphorylates dishevelled. EMBO J. 1997 Jun 2;16(11):3089-

96.

77

211. Wodarz, A., and R. Nusse. 1998. Mechanisms of Wnt signaling in

development. Annu. Rev. Cell Dev. Biol. 14:59–88.

212. Wong HC, Mao J, Nguyen JT, Srinivas S, Zhang W, Liu B, Li L, Wu

D, Zheng J. Structural basis of the recognition of the dishevelled DEP domain in the

Wnt signaling pathway. Nat Struct Biol. 2000 Dec;7(12):1178-84.

213. Wong HC, Bourdelas A, Krauss A, Lee HJ, Shao Y, Wu D, Mlodzik M,

Shi DL, Zheng J. Direct binding of the PDZ domain of Dishevelled to a conserved

internal sequence in the C-terminal region of Frizzled. Mol Cell. 2003

Nov;12(5):1251-60.

214. Xiao-qing Gan, Ji-yong Wang, Ying Xi, Zhi-li Wu, Yi-ping Li, and Lin

Li1. Nuclear Dvl, c-Jun, -catenin, and TCF form a complex leading to stabiLization

of -catenin–TCF interaction. J Cell Biol. 2008 March 24; 180(6): 1087–1100. doi:

10.1083/jcb.200710050.

215. Zeng, L., F. Fagotto, T. Zhang, W. Hsu, T.J. Vasicek, W.L. Perry, J.J.

Lee, S.M. Tilghman, B.M. Gumbiner, and F. Costantini. 1997. The mouse fused

locus encodes Axin, an inhibitor of the Wnt signaling pathway that regulates

embryonic axis formation. Cell. 90:181–192.

216. Zimmermann, K. W. (1898). Beitrage zur Kenntniss einiger Drusen und

Epithelien. Archiv fur Mikroskopische Anatomie 52, 552-706.

217. Zhang Y, Appleton BA, Wiesmann C, Lau T, Costa M, Hannoush RN,

Sidhu SS. Inhibition of Wnt signaling by Dishevelled PDZ peptides. Nat Chem Biol.

2009 Apr;5(4):217-9. Epub 2009 Mar 1.

218. Zhou, C.-J., Zhao, C. & Pleasure, S. J. Wnt signaling mutants have

decreased dentate granule cell production and radial glial scaffolding abnormalities.

J. Neurosci. 24, 121–126 (2004).