U
NIVERSITY OF
G
ENEVA
FACULTY OF MEDICINE DEPARTMENT OF NEUROSCIENCESK
AUNAS
U
NIVERSITY OF
M
EDICINE
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.
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……….…….…………222.1.6 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
3.1.3 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
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.
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;
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. 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.
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.
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;
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
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.
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
• 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
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.
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, inparticular 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.
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
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)
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.
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 C 2 and a rise in cytosolic Ca2+. Shah and colleagues applied those compounds to the ciliated cells. The results were induced a
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.
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 p120catenin 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
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.,
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).
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
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.
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.
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
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.
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
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
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 GSK3 102. 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.
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.
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
-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
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
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.
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.
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.
