Marco Natali Master Thesis

UNIVERSITA' DEGLI STUDI DI PISA

Facoltà di Scienze Matematiche, Fisiche e Naturali

Corso di Laurea Specialistica in Scienze e Tecnologie Biomolecolari

TESI DI LAUREAHow would it be possible to promote neural regeneration

in adult brain? A lesson from urodele amphibian

Anno Accademico 2009/2010

Relatori: Candidato:

Prof.ssa Renata Batistoni, Università di Pisa Marco Natali

Ph.Dott. Andras Simon, Karolinska Institutet Matricola: 301288

En los centros adultos las vías nerviosasson casi fijas, terminadas, inmutables.

Todo puede morir, nada puede ser regenerado.Queda para la ciencia futura cambiar,

si es posible, este severo decreto.

Santiago Ramon y Cajal, 1928

INDEX

Abstract1 Introduction1.1 Neural Regeneration in XX century1.2 Adult mammalian regeneration1.2.1 Study on stimulated neurogenesis1.3 Salamander adult regeneration1.4 Neurodegeneration: the Parkinson's Disease1.4.1 Treatments1.5 Aim of the thesis1.5.1 Gene candidates2 Materials and Methods2.1 Buffers preparation2.2 Animals2.3 6-OHDA injury2.4 Tissues harvesting2.5 In situ hybridization2.5.1 Probes2.6 Immunostaining2.7 Antibodies2.8 Mounting2.9 Microscope2.9.1 Image handling for pixel counting quantitative analysis2.9.2 Other microscope3 Results3.1 Optimisation of in situ protocol3.2 Primary screen of genes of interest3.3 nRAD3.4 Jar23.4.1 More detailed analysis of the role of JARID23.5 Notch4 Discussion4.1 Notch4.2 nRAD4.3 Jar24.4 ConclusionReferences

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I

Abstract

One of the medicine's dogmas in the last century assumed impossible to generate new neurons by an adult mammalian. However, in the second half of the century, active neurogenesis of Neural Stem Cells was found in some brain regions (subventricular zone and subgranular zone). Regenerative capabilities of neural structures in an adult organism are extended in other animal species, for example in urodele amphibians. Salamanders can regenerate various tissues and organs, it is hypothesized that salamander cells are able to de-differentiate in a stem-like condition and proliferate in order to rescue lost tissues.It is possible, thanks to neurotoxins such as 6-hydroxydopamine, to induce Parkinsonian-like injuries in salamander midbrain, and these animals can rescue the lesions in 30 days.The Parkinson's Disease (PD) is a disorder characterized by progressive neural degeneration with slow course, typical of aged people and by primary symptoms involving the motorial system. The causes of PD are not completely understood yet, and this increases the difficulty of studies aimed to fight the disorder and to discover a standard therapy. Various therapeutic approaches are thought out in order of reverting Parkinsonian neurodegeneration and repairing injured tissues, among these is the induction of adult neurogenesis at the midbrain level.Aim of my thesis work is to assess the involvement of some candidate genes in neurogenesis of adult newts (Nothophtalmus viridescens), in order to get an insight into the gene pattern that control regeneration in midbrain and to allow future comparative studies with mammals. This work was performed in Dr. Andras Simon's laboratory at CMB Department of Karolinska Institutet in Stockholm (Sweden) from January 2010 to October 2010.Expression of these genes were tested by in situ hybridization technique in regenerating salamander model with midbrain lesioned by 6-hydroxydopamine. Control samples were done with the sense probe of candidate gene in place of the antisense one. The in situ protocol needed to be set up and adapted to salamander brain slices before to work, so a probe for Sonic Hedgehog was used since its expression in newt was already known.- Notch, codifies for a receptor protein that suppresses neural differentiation and is thought to mantain cells in an undifferentiated state. This gene was chosen as marker of stem-like cells. Although results presented expression of Notch only in cells known to be stem-like, the control samples showed some colouration. It was hypothesized the presence in the tissues of the opposite strand of RNA. Probes for other genes did not show this phenomenon.- CKM, codifies for a creatine kinase involved in ATP production for energy supply in muscles, it has also an homologue in brain. This gene was chosen as negative control expected to not be expressed. Results do not show any

II

expression.- Annexin1, in mammals codifies for a membrane protein involved in anti-inflammatory processes in the Central Nervous System, so to preserve the CNS from neuropathologic worsening. This gene was chosen in order to understand if anti-inflammatory processes have an important role in regeneration of brain. Results did not show any expression.- Jarid2, in various species codifies for a transcription factor involved in epigenetic activation of developmental genes by Polycomb Complexes. This gene is known to be fundamental for differentiation of embryonic stem cells and of mouse neural embryonic development, so was the main candidate for this study. Results showed down-regulation in regenerating tissues and an indefinite localization.A further experiment, Jarid2 immunostaining, was also performed with a double aim: a) to compare the protein localization related with glial and neural cells; b) to check with a quantitative analysis if the down-regulation of Jarid2 during regeneration was effective. Results confirmed this hypothesis and showed a spread localization in neuronal cells, but a strong and definite expression in the stem-like cells.- RAD, in various species codifies for a small GTPase (variant of RAS) involved in muscle regeneration, so to be used as marker for regenerating muscles. This gene was chosen in order to compare regeneration between muscle and brain. Results show a strong expression in regenerating tissues strictly localized in few cells.As further experiment, the defined expression of RAD was compared with the presence of PCNA+ cells in order to understand if it was possible to exclude an eventual relationship between RAD and proliferation. The obtained result not confirms this possibility.

III

1 Introduction

1.1 Neural Regeneration in XX century

It was long thought that the mammalian brain was a static fixed structure:

"In the adult centers the nerve paths are something fixed, ended and

immutable. Everything may die, nothing may be regenerated. It is for the

science of the future to change, if possible, this harsh decree." (The Nobel

prize winner Ramon y Cajal, 1928)

This changed in the second part of '60s, when Joseph Altman used tritiated

thymidine to mark DNA and check mitotic activity. He discovered

constitutive proliferation in the hippocampus (Altman & Das, 1965) and

olfactory bulb (Altman, 1969) in rats. Scientific community did not accept

this discovery as prove of adult neurogenesis untill two decades after,

because was not possible to understand if neurons or other types of cells

were involved in this proliferation.

In '90s the evidence was found that precursor cells isolated from the

forebrain could differentiate in vitro into neurons (Reynolds and Weiss,

1992; Richards, 1992). In the following years, using immunofluorescent

labeling for bromodeoxyuridine (that labels DNA during the S phase of

proliferation) and for one of the neuronal markers, adult constitutive

neurogenesis was demonstrate in fishes, reptiles, birds, mammals and also

humans (Eriksson, 1998) injecting BrdU in cancer patients before death.

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1.2 Adult mammalian regeneration

Today, constitutive neurogenesis in adult human is known to involve only

the sub-ventricular zone of the olfactory bulb (SVZ) and the sub-granular

zone in dentate gyrus of the hippocampus (SGZ).

In the SGZ there are two cell types, identified as type 1 and 2, that form

new neurons with different frequency. While in SVZ there are two other cell

types, named type B and C, that present similar characteristic and form

new neurons.

Types 1 and B express glial fibrillary acidic protein (GFAP), they are

astrocytes and they divide infrequently. Instead types 2 and C does not

show glial characteristic and they divide continuously.

Type C cells are identified as transit amplifying cells (Belluzzi 2003), able

to give rise to immature neuroblasts.

Type B cells, localized inside the ependymal cell layer, are able to give rise

to type C cells when destroyed (Doetsch 1999), then they are identified as

adult neural stem cells of SVZ also if they has characteristics similar to

differentiated glia cells.

Other areas of the brain are generally considered non-neurogenetic,

although some studies suggest that low levels of neurogenesis may occur. In

2001 Gould observed new neurons BrdU positive in the white matter

(neocortex) of monkey, the possibility of migration from SVZ in the rostral

flux was considered (Gould 2001).

In 2003 it was reported that constitutive neurogenetic activity was present

in the mouse substantia nigra, and produced dopamingeric neurons.

2

Figure 1Schematic of progenitor types and lineages in the adult brain SVZ. NSCs in the wall of the lateral ventricles of adult rodents correspond to type B cells (SVZ astrocytes). These cells retain epithelial properties, including extension of a thin apical process that ends on the ventricle and a basal process ending on blood vessels. B cells give rise to C cells, which correspond to nIPCs. B cells also generate oligodendrocytes through oIPCs. Dashed arrows illustrate hypothetical modes of division: blue for asymmetric and red for symmetric divisions. Investigators do not currently know how many times C cells divide.SVZ, sub-ventricular zone;NSCs, neural stem cells;nIPCs, neurogenic intermediate progenitor cells;oIPCs, oligodendrocytic intermediate progenitor cells.

This activity also seemed to increase after ablation of dopaminergic

neurons (Evidence for neurogenesis in the adult mammalian substantia

nigra. Zhao 2003). However, the following year a different research group

published that there was no evidence for new dopaminergic neurons in the

adult mammalian substantia nigra (Frielingsdorf 2004) generating a

controversy on this matter.

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1.2.1 Study on stimulated neurogenesis

Promoting neural regeneration after an injury can be defined as to

stimulate neural stem cells to proliferate, migrate, differentiate and thus

lead to tissue repair. To be able to undergo neural regeneration would

means that there is a possibility to rescue adult brain tissue after an injury

and also to cause the regression of neurodegenerative diseases.

It is hard to study adult neurogenesis within humans with the current tools

available. In order to gain a better understanding of adult neurogenesis

without invasive study on adult human brain, animal models are used.

Mices and rats are the two most used mammalian model, the first to study

genetic correlations and linkage, the second to study drug response.

Although a lot of work has been in mammalian models of neurogenesis,

regeneration of complex structures, such as brain tissue has not been

achieved. In particular, we have controversial evidences of regeneration in

midbrain related to Parkinson's Disease (Zhao 2003; Frielingsdorf 2004) or

its induction due to neurotoxins.

Stimulating the proliferation of neural cells in mammalian adult forebrain

is possible. It was proven that Shh protein (Sonic Hedgehog), is involved in

various organogenetic processes. In forebrain it affects periventricular

GFAP+ astrocytes and GFAP- early precursors, and stimulates

proliferation raising the number of neurons. Moreover, blocking Shh

function results in decrease of the neurons' number (Sonic hedgehog

controls stem cell behavior in the postnatal and adult brain. Palma et al.

2004. Development).

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Also the neurotransmitter dopamine may also have a role in neurogenesis,

because the loss of dopaminergic neurons or blockage of dopamine

production, decrease the proliferation of neural stem cells both in sub-

ventricular zone and in sub-granular zone (Höglinger et al. 2004).

1.3 Salamander adult regeneration

Salamander's regeneration abilities include the tail (Iten 1976), limbs

(Benzo 1975), the heart (Bader 1978), ocular structures (Keele 1973) and

parts of their brain (Parish 2007). Salamander is an useful animal model in

order to study adult regeneration. The brain of the highly regenerative

salamander, the red spotted newt (Notophthalmus viridescens), in

physiological condition shows similar distribution of active germinal niches

with mammalians. Proliferation zones are essentially restricted to the

forebrain (telencephalon and rostral diencephalon's areas) of the newt

under normal homeostatic condition (Berg 2010).

Unlike mammalians, salamanders do not have ependymal cells, instead the

ventricles are lined by radial glia-like cells so called ependymoglial layer.

The ependymoglia shows stem proprieties and is GFAP positive (Benraiss

1996, Lazzari 1997).

The midbrain of the newt, after a parkinsonian injury induced by

neurotoxin, regenerate the lost tissue in 30 days (Parish, 2007).

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Figure 2Immunostaining of PCNA in adult salamander brain identifies constitutive proliferation zones. in telencephalon and rostral diencephalon.(A, A') longitudinal sections, schematic representation of the newt brain. (B-J) telencephalic transversal section. (B) No proliferation cells are detected in the rostral olfactory bulb (OB). (C) PCNA+ cells line the medial wall of the lateral ventricles in the accessory olfactory bulb.(D,E) Proliferating cells line the lateral walls of the lateral ventricle adjacent to the dorsal and lateral pallium (Dp and Lp, respectively). (F,G) Ventral accumulation of proliferation situated ventrally to the striatum (Str) in the region of the bed nucleus of the stria terminalis (Bst). (H) A ventrally located proliferation zone in the area of suprachiasmatic nucleus (Sc), and a medial zone situated adjacent to the ventral thalamic nucleus (Vtn). In the most caudal region of the lateral ventricles, proliferating cells are scattered on both the medial and lateral wall. Note unspecific cytoplasmic labelling between medial and ventral proliferation zone (arrowhead). (I,J) Lack of proliferating cells in the midbrain and hindbrain, respectively. Tm, midbrain tegmentum; Tc, tectum; LDT, laterodorsal tegmental nucleus; Ra, Raphe nuclei. Scale bar: 100 µm

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Figure 3ATime course of TH+ cell regeneration following 6-OHDA injection. Sham, n=4-14 animals per group per time point (d3, n=4; d5, n=6, d7, n=5; d10, n=6; d17, n=5; d24, n=4; d30, n=14). Lesioned, n=5-18 animals per group per time point (d3, n=5; d5, n=6; d7, n=5; d10, n=6; d17, n=5; d24, n=6; d30, n=18). Mean±s.e.m.; Student’s t-test; *, P<0.05; **, P<0.01.(Parish 2007)

Figure 3BTotal number of BrdU+ TH+ cells following lesioning. Sham-lesioned animals pulsed days 0-30, n=11; lesioned animals pulsed days 0-30, n=8. Lesioned animals pulsed days 0-3, n=8. Lesioned animals pulsed days 4-23, n=8. Mean±s.e.m.; ANOVA with Tukey post-hoc test; ***, P<0.001.(Parish 2007)

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1.4 Neurodegeneration: the Parkinson's Disease

One of the most common diseases characterized by neurodegeneration is

the Parkinsons's Disease, which affects about 2% of human population over

65 years old. Parkinson's Disease is characterized by three classical

symptoms: resting tremors, rigidity, hypokinesia; but later it leads to

disturbances in gait, balance and sometimes also automic disturbances,

dementia, depression. The rest tremor is maximal when the limb is at rest

and disappears during voluntary movements and sleep. Rigidity comes

from joint stiffness and increased muscle tone and is often related to pain.

The muscle is passively shaken and it is due to the loss of regulation

between flexor muscles and antagonist muscles (Jankovic 2008).

Figure 4Human midbrain samples: parkinsonian on the left, normal on the right.Visible reduction of the substantia nigra in the parkinsonian sample.

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In normal conditions gamma circuit feels muscle tone and the

extrapyramidal circuit (composed by basal nuclei) regulates its activity. In

Parkinson's Disease this regulation is lost and the subsequent increase of

muscle tone causes movement problems.

Hypokinesia can develop at different severity level, slowness of movements

called bradykinesia, or in the worst cases absence of movement called

akinesia, such as freezing episodes. This problem comes from the planning

and initiation of the movements, more than from its execution. Sometimes

immobile Parkinsonian patients are capable to perform rapid movements if

they get excited. This suggests that their motor programmes could be

intact, but patients have difficulties in controlling them.

At histopathological level, Parkinson's Disease is characterized by a

progressive loss of midbrain substantia nigra dopaminergic neurons that

project to the striatum. This loss results in a disequilibrium between

excitatory and inhibitory mechanisms which control movements, so it is

responsible for the main motor symptoms in the disease. In healthy brains,

dopamine released by dopaminergic neurons in striatum regulates the

activity of GABAergic neurons, these neurons then release GABA in order

to inhibit motorial neurons. In Parkinson's Disease, dopaminergic neurons

die and thus dopamine levels in the striatum drop, causing deregulation of

GABAergic neurons, which leads to motor symptoms typical of the disease.

Another histological feature of Parkinson's Disease is the presence of Lewy

neurites and Lewy bodies within neurons. Lewy neurites are inclusions

composed of abnormally phosphorylated neurofilaments, while Lewy bodies

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Figure 5Organization of the basal ganglia. The cortex projects to the striatum that is GABAergic and project to the Gpi and to the SNr (both GABAergic). Gpi and SNr are efferent and project to the thalamus that is excitatory on the cortex. The striatum also projects to the Gpe (GABAergic) that project to the subtalamic nucleus (glutaminergic), projecting to the efferent nuclei.Gpe = external globus pallidus, Gpi = internal globus pallidus, SNc = substantia nigra pars compacta, SNr = substantia nigra pars reticolata, STN = subthalamic nucleus, PPN = pedunculopontine nucleus; Thalamus: CM = centromedian nucleus, VA = ventral anterior nucleus, VL = ventral lateral nucleus

are aggregates of proteins containing misfolded alpha-synuclein fibrils

together with other proteins such as ubiquitin. The reason why cells fail to

eliminate the misfolded alpha-synuclein through ubiquitination and

proteasome recycling is not known. These histological features appear

before any early symptoms of Parkinson's Disease and before any neuronal

loss has occurred, but unfortunately there is no diagnosis can be done

without an autopsy.

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The cause of Parkinson's Disease is largely unknown, because the majority

of cases are sporadic, however, several implied genes and susceptibility

factors have been identified in the last decade. According to these studies,

an abnormal increased oxidative stress and mitochondrial dysfunction,

together with protein misfolding and impairments in the ubiquitin-

proteasome and autophagy-lysosomal systems, contribute to Parkinson's

Disease.

Primary symptoms of Parkinson's Disease can be also induced by

neurotoxins: in 1980s some Californian heroin addicts used a drug and

developed symptoms of Parkinson's Disease.

The drug was MPPP, desmethylprodine similar to morphine, but its

synthesis under improper condition produced MPTP as a major product.

MPTP can pass the hematoencephalic barrier and, and once in the glial

cells, it is converted into MPP+ that can be transferred by dopamine

transporter inside dopaminergic neurons, where it blocks mitochondrial

NADH dehydrogenase leading neuronal death (Chiueh 2006).

1.4.1 Treatments

A common therapy of Parkinson’s disease is to artificially increase the

dopamine levels caused by the loss of the dopamingeric neurons. This can

be done through administering Levo-Dopa, a precursor of dopamine that

can cross the hematoencephalic barrier. However this treatment is only

useful to temporary stop motor symptoms of Parkinson's Disease, and it can

not stop the neural degeneration. Moreover patients cannot take L-Dopa for

long periods because this induces dyskinesias, another motorial disorder

(Simuni 1999).11

There is a surgical treatment, based on the inserting electrodes to stimulate

the internal segment of globus pallidus, the subthalamic nucleus or the

pedunculopontine nucleus (Silberstein, 2002). A device send continuously

electrical pulses to the target area inside the brain, this obtain to block the

impulses that cause tremors in a reversible way respect to a surgical

elimination of the brain areas involved.

However this is just a symptomatic therapy, it cannot stop the further

neuronal degeneration and have no effect on secondary symptoms of

advanced Parkinson's Disease.

The immunity system can be involved in degeneration of parkinsonian

neural tissue (Qian 2010). Microglia are considered to be the Central

Nervous System counterparts of peripheral macrophages, they quickly

respond to immunological stimuli with a burst of production of pro-

inflammatory mediators. An excess of these pro-inflammatory mediators

can aggravate the neuropathology and neuronal death, so other factors are

needed to regulate inflammatory response and decrease neural

degeneration.

Another method to increase dopamine levels within the striatum is based

on cell therapy. This strategy requires harvesting fetal or embyonic stem

and progenitor cells, differentiating them into dopaminergic neurons,

grafting these neurons into the striatum of patients and integrate them in

order to rebuild the lost connections.

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Some problems can rise with this treatment. In patients that used L-Dopa

for long time, dyskinesias get worse with the graft (Hagell 2002). In

patients that suspend immunosuppression, the innate immune system is

newly activated and functional improvement of the graft disappear.

However recent studies have shown that grafted neurons survive at

maximum as long as 16 years in brain of parkinsonian patients after

transplantation (Mendez 2008).

There are two source of cells for these experiments, from cell lines and from

embryos, but in humans only cells directly isolated from the embryos have

been used.

The fetus is known to be a great source of cells so it is not strange to use the

same mesencephalic tissue of embryo as a pool in order to harvest cells for

the graft. However there are some problems associated with this method.

First of all ethical issues that limit the availability of the tissue according

to quantity, quality and abortion time, so it is not simple to obtain the

correct cells for the graft. Moreover, the survival of fetal tissue after

transplantation seems to be less than 10% in the best of the cases

(Sortwell 2007).

It is possible to induct stem cells to differentiate in dopaminergic neurons

thanks to transcription factors, morphogens and survival factors, according

to defined protocols (Zhang 2010).

This strategy is based on understanding the development of midbrain

dopaminergic neurons and the ability to identify dopaminergic

differentiation factors.

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The alternative to cell replacement therapy would be to stimulate

endogenous neurogenesis. Neurogenesis could help to stop degenerative

processes by replacing the lost tissue.

1.5 Aim of the Thesis

This work characterizes itself as a study about involvement of the genes in

regulation of adult neurogenesis, focusing on a non-mammalian vertebrate

model, the red spotted newt, because its extensive regeneration abilities.

The chosen model is the newt with midbrain injured by 6-OHDA

neurotoxin, that eliminates dopaminergic neurons and mimes parkinsonian

symptoms.

1.5.1 Gene candidates

In order to promote neural regeneration, it must be understood the process.

To check the variation of expression and localization of candidate genes

after an injury, allows to understand which genes are implied in promoting

neural regeneration, to study neurogenic processes and to test the

regenerative potential of the vertebrate brain.

Recently the transcription profile of regenerating cells was analyzed thanks

to a micro array work (Berg, 2010) and from this list several candidate

genes that could have significant roles in the regeneration process were

identified.

Three different genes were chosen as candidates to start this study: AnxA1,

JARID2, nRAD; and two more genes, CKM and Notch, were chosen

respectively as negative control and stem marker.

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Notch encodes a receptor protein that participates in many developmental

cell fate decisions. Notch suppresses neuronal differentiation and is thought

to maintain precursor cell properties. In postnatal mammalian sub-

ventricular zone cells, Notch prevents migration to the olfactory bulb,

suppresses neuronal differentiation and decreases proliferation (Chambers,

2001).

Notch is involved in a lot of different processes and tissues, but in each case

its role seems to be related to the maintenance of stemness.

CKM (Creatine kinase of muscle) is expressed in skeletal muscle, in heart

and in vessels, and it has an homologue expressed in brain. Its role is

related to energy supply for cells and there are no evident reasons to relate

its role with neurogenesis, so it was chosen as negative control.

AnxA1 encodes a protein, Annexin A1 or Lipocortin 1, which is associated

with many cellular components, including plasma membrane

phospholipids, vesicles and cytoskeletal proteins. This protein plays a role

in differentiation, proliferation, plasma membrane repair, apoptosis and

several anti-inflammatory processes out of the cell (McKanna 1995; Solito

1998; Lim 1998; de Coupade 2000; Solito 2001; McNeil 2006).

In mammals AnxA1 appears to be constitutively expressed in the cells of

the innate immune system of the normal brain. Microglia are considered to

be the Central Nervous System counterparts of peripheral macrophages,

they rapidly respond to immunological stimuli with a burst of production of

pro-inflammatory mediators and are capable of phagocytosis.

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An excess of these pro-inflammatory mediators can aggravate the

neuropathology and neuronal death (Carey 1990; Mogi 1994; Muller 1998).

Annexin A1 inhibits several of these factors and suppresses the activity of

polymorphonucleates and macrophages, regulating in this way the

inflammatory response and promoting neuroprotection (Solito 2008).

Studying AnxA1 in Parkinson's Disease models allows to understand the

role of neuroprotection as rescue mechanism for degenerating brain tissue.

JARID2 (Jar2) encodes a transcription factor of the ARID family (AT rich

interaction domain), involved in several biological processes, also called

Jumonji. The Jumonji name, japanese word for "cruciform", derives from

the mutant mice jar2-/- that showed an abnormal brain morphology at fetal

stage E13.5 (Takahashi 2004).

JARID2 is part of PRC2 (polycomb repressive complex 2), a complex that

regulates developmental genes expression pattern in embryonic stem cells

and also during development. PRC2 contains three core subunits, all of

which are necessary for trimethylation of lysine 27 on histone 3

(H3K27met3) in order to silence gene expression. The role of JARID2 is

identified in recognition of DNA sequences and recruitment of the complex.

In xenopus, JARID2 depletion results in aberrant increase of H3K27

methylations, failure of differentiation and developmental gene induction,

so that development stops at gastrulation (Peng & Wysocka 2009).

In mouse embryonal stem cells, Jar2 depletion results in failure of

trascriptional activation related to differentiation marker genes and

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abnormal epigenetic pattern (Pasini & Helin 2010).

In mammalian heart, JARID2 binds to the promoter of cyclin D1 and

represses the transcriptional activity in order to suppress cardiac myocyte

proliferation. However its role is closely related with cyclin D1 and cell

cycle exit, also in brain tissues. During neurogenesis in particular, JARID2

seems to be involved in neuronal differentiation and migration of neural

progenitor cells (Takahashi et Takeuchi 2007).

RAD (Ras associated with diabetes) is a variation of a RAS signaling small

GTPase expressed in skeletal muscle of diabetic patients, and constitutively

in hearth and lung (Reynet et Kahn 1993).

RAD interacts with calmodulin, protein kinase II (Moyers et Bilan 1997)

and betatropomyosin (Zhu et Bilan 1996) and it seems to play a role in

muscle regeneration, but its role is not clear yet. In rats, RAD is found to be

expressed in vascular smooth cells where there is a vascular lesion

(Fu et Zhang 2005).

In newt, nRAD is expressed in skeletal muscle cells within 4 hours after

limb amputation. The presence of retinoic acid, that causes an enhanced de-

differentation of myotubes in regenerating limbs, can increase the

expression of nRAD after amputation

(Shimuzu-Nishikawa, Tsuji et Yoshizato 2001).

nRAD is than used as marker for regeneration in muscle and its presence

as gene candidate has the aim to discover an eventual parallelism between

brain and muscle regeneration.

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2 Materials and Methods

2.1 Buffers preparation

0.2 M phosphate buffer

165.3 g Na2HPO4 x 7H2O (268.07 Da)

25.6 g NaH2PO4 x H2O (137.99 Da)

Water to 4 liters

PBS

2 liters 0.2 M phosphate buffer

35 g NaCl

Water to 4 liters

4% Paraformaldehyde

200 ml water

16 g PFA

Heat to 65°C and check pH between 7.2 and 7.4 adding 10M NaOH

200 ml 0.2M phosphate buffer

3 g NaCl

Filter to 0.2 µm

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Prehybridization Buffer

10 ml deionized formamide

5 ml 20x SSC

2 ml 50x Denhardt's

5 mg yeast RNA (sigma R6759)

10 mg salmon sperm DNA

Water to 20 ml

B1

100 ml 1 M Tris-HCl pH 7.5

30 ml 5 M NaCl

Water to 1 liter

B3

100 ml 1 M Tris-HCl pH 9.5

20 ml 5 M NaCl

10 ml 5 M MgCl2

Water to 1 liter

Filter to 0.45 µm

B4

4.5 µl (NBT) Nitroblue Tetrazolium (Boehringer 1 383 213)

3.5 µl (BCIP) 5-Br-4-Cl-3-indolylphosphate (Boehringer 1 383 221)

0.24 µg Levamisole

B3 to 1 ml

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2.2 Animals

Adult red spotted newts, Notophtalmus viridescens (Charles Sullivan Co.,

Nashville TN), were mantained in a humified room at 25°C and fed weekly.

All experiments were performed according to European Community and

Local ethics committee guidelines.

2.3 6-OHDA injury

Newts were anaesthetized by placing them in an aqueous solution of 0.1%

Tricane for 20 minutes. Animals were placed in a neonatal stereotaxic head

frame. 200 nl of a solution consisting in 6-OHDA (6 µg/µl) and ascorbic acid

(0.2 mg/ml) was injected into the third ventricle with a glass micropipette

through a small hole drilled in the skull.

Subsequently the surgery hole in the skull was sealed with dental cement

and animals were left to recover overnight in a shallow container of water

before being placed back into a 25°C water environment.

2.4 Tissues harvesting

Animals were anaesthetized by immersion in an aqueous solution of 0.1%

Tricane for 20 minutes and perfused with 4% formaldehyde in PBS.

Animals were dissected and brains were rapidly placed in 4%

formaldehyde. After 1 hour of post fixation, brains were cryo protected in

20% sucrose in PBS for 12 hours and then embedded in OCT compound.

16 µm coronal sections were collected alternating on seven slides and stored

at -80°C.

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2.5 In situ hybridization

In situ allow to study the macroscopic distribution and cellular localization

of DNA and RNA sequences in a heterogeneous cell population. In this

study all the protocol was set to put in evidence RNA localization, so to

understand the expression of candidate genes.

Two digoxigenin-marked RNA probes were sinthetized for each candidate

gene, one sense and one antisense. The antisense probe annealates

specifically with mRNA of the gene during hybridization, so its expression

is revealed; while the sense probe has not any mRNA counterpart and

cannot annealate specifically, so nothing else than background is revealed

and this sample could be used as reference.

The first part of in situ technique require an RNAse free enviroment to

avoid the destruction of RNA strands.

Brain slices were completely fixated in 4% formaldehyde in PBS for 10

minutes, quickly washed in PBS and than permealized in a buffer 1.333%

triethanolamine, 0.175% HCl, 0.25% acetic anhydride in DEPC-treated

water for 15 minutes.

Slices were washed 3 times in PBS and treated with Prehybridization

Buffer at 37°C for 2 hours in a chamber humified with a solution of 50%

formamide and 5x SSC, than heat-denaturated RNA probe was added at

Prehybridization Buffer, slides were covered and let O/N at 70°C in the

humid chamber. Once the hybridization was done, the RNA strands can not

be attacked by RNAses.

Coverslips were removed and slides were put in 0.2x SSC at 70°C for 2

21

hours, than cooled at room temperature before to prepare slices with B1.

Slices were treated with B1 containing 10% blocking reagent for 1 hour in a

chamber humified with B1 itself, than 1:5000 Anti-digoxigenin antibody

coniugated with alcaline phosphatase enzyme was added to B1 and slices

let O/N at 4°C.

Slices were washed with B1 and equilibrated with B3, before to treat them

with B4 reagent and let it work at room temperature in a chamber

humified with B3 for a long time (from 2 hours to 2 days) untill the dye

produced by the alcaline phospatase antibody-coniugated marked tissue

visibly.

Slides were than washed in PBS to stop reaction and to reduce background

signal, then mounted in mounting media (DAKO) added with DAPI.

2.5.1 Probes

Clones of E. coli inserted with plasmid (pCR-BLUNT II-TOPO) for the

required gene sequence, stored at -80°C were thawed out and let to grow in

Luria-Bertani broth medium over night. The DNA was purified from the

growth thanks to MiniPrep kit by Qiagen and than digested to linearize the

plasmid.

Digestion with BamH1 restriction enzyme cuts the plasmid on the target

sequence of SP6 primer, meanwhile digestion with XHO1 cuts the plasmid

on the target sequence of T7 primer.

Figure 6Gel to check linearization of the plasmid.L = ladder; SP6 = plasmid digested by XHO1;T7 = plasmid digested by BamH1; C = control

22

Figure 7Map of the plasmid pCR-Blunt II-TOPO

Digested DNA was purified with Qiagen spinning column and its

concentration checked with NanoDrop 2000 spectrophotometer (Thermo

scientific).

RNA was synthesized from this DNA template using NTPs coniugated with

digoxigenin as marker, and treated with DNAse.

23

Sequence of Newt-Shh

1 AGTGGCCCTG GAGTCAAGCT GCGGGTGACC GAGGGTTGGG

41 ATGAGGATGG CCACCACTCT GAGGAGTCCC TGCACTACGA

81 GGGTCGGGCA GTGGACATCA CCACCTCGGA CCGGGACCGC

121 AGCAAGTATG GCATGCTGGC CCGCCTGGCC GCGGAGGCTG

161 GCTTCGACTG GGTCTACTTT GAGTCCAAGG CCCACATCCA

201 CTGCTCAGTG AAAGCAGAGA ACTCGGTAGC TGCAAAATCG

241 GGAGGCTGCT TCCCAGGCTC TGCCACGGTG ACCCTGGAGA

281 AAGGCATAAG GATGCCAGTG AAGGACCTGA GGCCGGGGGA

321 CAGGGTGCTC GCCGCAGATG GACAGGGCCG GCTGGTCTAC

361 AGCGACTTCC TCTTGTTCAT GGATAAAGAG GCGACGGCCA

401 GGAAAGTCTT CTACGTGATA GAGACCTCTC AGCCCCGGGA

441 GAGGCTCCGC CTGACCGCCG CTCACCTCCT CTTCGTAGCC

481 CAGGCGCACC CAGGAAACGC CAGTGGGGGC AACTTCCGGT

521 CCATGTTTGG CAGCGCAGGC TTCCGCTCCA TGTTCGCCAG

561 CAGCGTGCGG CCGGGGCACC GGGTGCTCAC AATGGACCGG

601 GAAGGCCGGG GGCTAANGGG AAGCCACAGT GGAGCGAGTG

641 TACCTGGAGG AGGCCACGGG GGCCTACGCT CCCGTCACTG

681 CACACGGGAC CGTGGTCATA GACAGGATGC TGGCCTCCTG

721 CTACGCGGTC ATAGAGGAAC ACAGGTGGGC ACACTGGGCC

761 TTCGCCCCTC TGAGAGTGGG CTTCGGGGTC TTGTCTTTCT

801 TCTCCCCCC

24

Sequence of Notch

1 TGACCAATCG GAGAAGTTGG AATGAGGAGA AGAGCTCGAC

41 CATTGGTCTG GGGATTCAGG AGATGGAGTC AAAAAGGGGT

81 GATCAGACAC CTGAAGTTGG TGGCTGGGAG TGTTGTCCAT

121 TGGAGAAGAG TAGCTATGCT GAGAAGGCGG

Sequence of CKM

1 CTTCCTGGTC TGGGTCAACG AGGAGGATCA TCTCCGTGTC

41 ATCTCTATGC AGAAGGGCGG CAACATGAAG GAGGTGTTCA

81 GGCGCTTCTG TGTTGGATTG CAGAAGATTG AAGAGATCTT

121 CAAGAAAGCT GGCCATCCCT TCATGTGGAA CGAGCATCTT

161 GGTTACGTGT TGACCTGCCC CTCCAACTTG GGCACTGGAC

201 TACGTGGTGG TGTCCACGTC AAACTCCCCA ACCTCTGCAA

241 GCACCCCAAG TTTGAGGAAA TTCTGACCAG ACTGCGCCTA

281 CAGAAACGTG GTACAGGTGG TGTGGATACC GCAGCTGTTG

321 GTGGTGTCTT TGACATCTCA AATGCTGACC GTCTGGGGTT

361 CTCTGAGGTA GAGCAGACAC AGATGGTGGT GGATGGCGTG

401 AAGCTCATGA TTG

25

Sequence of Jar2

1 GATTTCCTTA CGTTTCTATG TCTTCGAGGT TCTCCAGCCT

41 TGCCCAGCTC CATTGCGTGT CTTGGAACCT CGCTAGATGA

81 AGACGACGTG GAGGAGGAGG AGGATGAAAC ATGTTCTCAA

121 CGGACATGTG TTTAATGGGT ACAGCAAATC ACCAAGGGAA

161 AAAGAATCCG CCCAGAAACA CAAAAGTAAA GAAGCCACAC

201 CAGGGAAGGA GCGGAATGCT GAACAGAGGG TTGAGAGGCG

241 AAGAGAGCGG GCCGCTGCTG CTGCTAACCA CACTCCTGCT

281 CCACATACTG TCTCCTCCGC CAAAGGTCTC GCTGCTAGCC

321 ACCATACACT TCACAGATCG GCTCAGGACT TAAGGAAA

Sequence of AnxA1

1 TAGTCTCCTT TGGTTTCATC CAAAATAGCC TGGCGGAGTG

41 ATATGCCATA CATTCGCTTG TAATGTGCCT TGATCTCATC

81 CATATCAATT CCGGAACGGG AAACCATGAT TCTGATTAAT

121 TGCTTGTCAC GAGTTCCAGA TCCCTTCATA GACAAGAATA

161 GTCGTTCAGC AAAGTAAGAT GGCTTGCTTA CTGCACATTT

201 CACAATAGCG GTCAGGCATT TTTCAATATC ACCCTTCAGC

241 TCAAGATCCA GGGCCTTGTT CATGTCATGT TTACTGTAGC

281 TGGTATACCT CTGAAAGACC TTACGAAGGT GTGGACCACT

321 TCTTGTAGTA AGAAGGTCGA TGAACACCTT AACATCGGTT

361 CCTTTTCTCT TTTCTCCAGC TTCGTA

26

Sequence of nRAD

1 GCGTTGTCTT CTTTGCTGTC CTTGCGGAGT CGGATCTGTC

41 GGACGATGCC CTCAAACAGG TCCTTAACGT TATGGTGAAG

81 GGCGGCTGAC GTCTCGATGA ACTTGCAGTC AAACACTACC

121 GCGCATGCGC GGCCTTCTTC CACAGACACT TCTCGTGATC

161 TCACCAGATC ACTCTTGTTC CCCACGAGGA TGATTGGAAT

201 GTCTTCGCTC TGCCGCGCTC TCCTCGGCTG TATCCTCAGC

241 TCCGAGGCCT TCTCAAAGCT GGACTTGTCC GTCACAGAGT

261 ACACAATGAC GTATGCATCC CCCATTTTCA TGCACTGATT

301 CTGAAGCCAG TGGGTCTCAT CCTGCTCCCA TATGTCATA

2.6 Immunostaining

Slides with brain slices were fixed in 4% PFA for 5 minutes at room

temperature and then rapidly washed in PBS. Tissue slices were

permeabilized with 0.2% Triton-X in PBS for 15 minutes on a rocker and

then quickly washed again in PBS.

Slices were protected by aspecific antibodies binding in blocking serum (5%

goat serum, 0.2% Triton-X, PBS) for 1 hour at room temperature and than

primary antibodies were substituted to serum and let bind for 2 hours.

After primary antibodies binding, tissue slices were washed in PBS 3 times

and secondary antibodies fluorecrome-coniugated were placed on slides and

incubated for 2 hours at room temperature.

After secondary antibodies binding, slices were washed in PBS 3 times and

mounted in mounting media (DAKO) added with DAPI.

27

2.7 Antibodies

rabbit anti-JARID2 (1:2000, AbCam)

mouse anti-GFAP (1:1000, Chemicon)

mouse anti-PCNA (1:800, Chemicon)

mouse anti-Neun (1:200, AbCam)

Alexa 448 goat anti-mouse (1:500, Molecular Probes)

Alexa 594 goat anti-rabbit (1:500, Molecular Probes)

2.8 Mounting

After both in situ hybridization or immunostaining, slides were quickly

washed in pure water at room temperature before to be mounted with Dako

Flourescent Mounting Medium added with DAPI, and fixed by coverslips.

2.9 Microscope

Image capturing was performed using "Microscope Axioplan 2 imaging"

linked with "Axiocam HRm" (Carl Zeiss Microscopy) and the Apple

executable "OpenLab".

All quantitative analysis were based on pictures from this microscope.

2.9.1 Image handling for pixel counting quantitative analysis

Image handling was perfomed by "gIMP image editor 2.6" working on RGB

channels.

For every picture of immunofluorescence, was established an highlight

defined area thanks to the command “Colors > Threshold”.

28

29

The “Threshold” effect equalizes the measured brightness, so to make

impossible recognize down-regulation and up-regulation inside cells, but

put in evidence if the signal is present or not.

Pictures of the marker to quantify, were then merged on DAPI pictures

related to the same sample in order to show the area of co-expression of the

two signals, quantifiable in number of pixels thanks to the command

“Colors > Info > Histogram”.

The ratio between the co-expression area and the total DAPI area is an

approximation of the relative number of marked cells.

2.9.2 Other Microscope

Other pictures for in situ hybridization samples were captured later in the

University of Pisa by "Microscope Nikon Eclipse DSU2" linked with "Nikon

Digital Sight" camera.

3 Results

3.1 Optimization of in situ protocol

The In Situ Hybridization technique was chosen to determine the

expression pattern of candidate genes and to localize it cell by cell in the

sample tissues.

Because Sonic Hedgehog (Shh) is an important gene also involved in

forebrain neurogenesis and its expression in salamander midbrain was

already observed in the same lab (Berg 2010), a probe for Shh was initially

used in order to establish a correct protocol for in situ hybridization. The

known expression of Shh could be used as reference to chose the better

condition of work.

Figure 8In situ hybridization of Shh antisense probe in adult salamander midbrain.(A) uninjured, (B) 7 days after 6-OHDA injection.(Berg, 2010)

30

The first attempt to optimize the in situ protocol was to decrease the time of

reaction for the production of the dye signal, in order to block

overproduction. The kinetic in production of dye seemed to be the same for

specific signal and background, because using different time of reaction

affected all the coloration in the same way. This showed that saturation

was not responsible of the background signal.

The second attempt to optimize the protocol was to increase the annealing

temperature to hybridize RNA, in the hypothesis of an unspecific probe

binding. Annealing temperature was set at 80°C, but no differences were

evidenced comparing the result to the one of the usual protocol.

An alternative explanation for this background excess could be due to an

unspecific antibody binding. To monitor this, it was preformed the in situ

hybridization bypassing some step of the protocol, so to expect as result no

signal at all, when the unspecific antibody is eliminated from the protocol,

or only background, when the unspecific antibody binds unsought epitopes.

In the in situ experiment without the Shh probe, as in the negative control,

no hybridization could happen and the primary antibody could not be bound

to the probe, but washed away or bound in unspecific way.

The result showed the same background signal already seen in previous

experiments and confirmed that background was not due to a lack of probe

specificity.

In the in situ hybridization where the use of primary antibody was

bypassed, secondary antibody could not be bound to the primary one, but

washed away or bound in unspecific way.

31

The result revealed that background was present and put in evidence that

primary antibody was not responsible of any lack of specificity.

In the in situ hybridization without the use of secondary antibody, alkaline

phosphatase could not be present in the sample, so the reaction of dye

production could not be catalyzed.

The result showed that background signal was still present and underlined

that coloration was not produced by alkaline phosphatase enzyme

conjugated with secondary antibody.

It was then hypothesized the presence of an endogenous enzyme activity in

the tissue that could react with the substrate of reaction and catalyze it, so

an in situ hybridization with regular protocol was repeated adding an

inhibitor (Levamisole) and the background signal was eliminated.

3.2 Primary screen of genes of interest

Each candidate gene was shown in a micro array experiment (Berg, 2010)

to be differently expressed in lesioned tissues comparing with control.

nRAD and AnxA1 appeared up-regulated in 6-OHDA injured animals,

while JARID2 appeared down-regulated.

To confirm this data and to localize the gene expression, in situ

hybridizations were performed on gene fragments that had been cloned.

The first aim was to determine if these cloned fragments were sufficient to

produce functional probes. Sense and antisense probes were prepared for

every gene fragment and checked on injured material, harvested 7 days

after the 6-OHDA injection.

32

While it was not possible to see any signal for CKM and AnxA1 probes,

indicating probably they did not work; Notch, nRAD and Jar2 showed some

signal localized around the 3rd ventricle. The working probes were

analyzed further comparing expression between injured and control

material.

3.3 nRAD

In situ was done for the candidate gene nRAD, in order to find eventual

correlation between muscular and neuronal regeneration.

The result put in evidence strong expression strictly localized in ventral

layer of ependymoglial cells in the 3rd ventricle of injuried animals. Then a

parallelism between muscolar and neuronal regeneration could be possible.

The signal marks a little number of cells close each other and disappears at

short distance along the rostro-caudal axis.

Figure 9In situ hybridization of nRAD antisense probe in adult salamander midbrain

33

With the purpose to see if RAD positive cells are proliferating cells, an

other experiment was set up. It was supposed the population of cells in two

consecutive slices from the same brain to be similar, since every slice was

16-20 µm thin. Slides were numbered, in situ for RAD was performed on

slides with even numbers, while immunostaining for PCNA was done on

slides with odd numbers.

The result shows the expression of RAD in injured tissues on the ventral

diencephalic part of the third ventricle, close to PCNA expression.

34

Figure 10Consecutive slices treated with different techniques in order to show the localization of PCNA and nRAD.

In particular, nor PCNA nor RAD were present 3 days after injection; 7

days after injection the expression of RAD was strictly localized in the cells

edging the third ventricle and the expression of PCNA was localized in

some cells spread around; 14 days after injection the expression of RAD was

disappeared, instead PCNA positive cells were diminished in number.

The similarity among two slides is not enough to demonstrate that RAD

positive cells are proliferative, but the result suggests a possible correlation

in the regenerative path between PCNA and RAD.

3.4 Jar2

For the candidate gene JARID2 were set up both an in situ hybridization

and an immunostaining, in order to check the expression level of Jar2

mRNA and protein JARID2, since the role of this gene in epigenetic

regulation identifies it as an important candidate to study differentiation

processes.

In situ result showed an evident signal in uninjured antisense-hybridized

sample and a light decreasing of signal in lesioned tissue, both localized

close to the ventricle cell layer.

This result indicate the repressive and anti-proliferating activity of Jar2

has to be down-regulated during regeneration in order to allow proliferation

and subsequent neural differentiation.

Immunostaining result showed a strong expression in the cells bordering

the third ventricle, and some other isolated cells with lower expression.

35

Figure 11In situ hybridization of Jar2 antisense probe in adult salamander midbrain

This distribution appeared similar between control and injured tissues.

Cause the variable expression of JARID2 protein in cells not edging the

third ventricle, next it was sought to determine which cells expressed

JARID2 in the midbrain.

It was performed some co-immunostaining with cell type specific markers:

one for GFAP and one for NeuN.

GFAP marks ependymoglia cells, which are stem cells in salamander brain,

while NeuN is a pan-neuronal marker.

The immunostaining result for JARID2 and GFAP double labelling

revealed co-localization in the cell layer bordering the third ventricle. All

the cells with strong expression of JARID2 were GFAP positive, and all cell

bodies of GFAP positive cells showed a strong expression of JARID2.

Double immunostaining result for JARID2 and NeuN did not show any

36

Figure 12Co-immunostaining for JARID2 and GFAP in adult salamander midbrain.(up) Uninjured, (down) 7 days after 6-OHDA injection.

37

Figure 13Co-immunostaining for JARID2 and NeuN in adult salamander midbrain.(up) Uninjured, (down) 7 days after 6-OHDA injection.

38

overt correlation. NeuN positive cells appear to be reduced in the injured

tissue, due to the neuronal loss induced by 6-OHDA.

3.4.1 More detailed analysis of the role of JARID2

A detailed quantitative analysis was performed in order to check if JARID2

protein expression presented differences between control and injured

samples: pictures of immunofluorescence were edited to individuate

highlight defined area of signals expression and gain a ratio.

A definite threshold was set for each fluorescent signal, and all pixels under

that value were not considered to individuate the area of expression as

signal.

JARID2 expression signal was considered only when co-localized with DAPI

signal in order to isolate background from cell expression.

Figure 14Picture handled for the quantitative analysis. Down the third ventricle.(blue) DAPI = nuclea; (purple) co-localization JARID2-DAPI = Jar2+ cells

39

Areas were calculated with pixel counting, and the ratio between JARID2

area and DAPI area represents a good approximation about which cells are

JARID2 positive.

Results show a small but significative decrease of JARID2 expression

respect to DAPI (P=3.44%) in the injured sample. This result seems to

confirm the one of in situ hybridization about down-regulation of Jar2

during adult brain regeneration.

Figure 15Percentual of JARID2+ cells in adult salamander midbrain.(left) Uninjured, (right) 7 days after 6-OHDA injectionMu=0.660 Dev.st.u=0.187 nu=14M7=0.555 Dev.st.7=0.201 n7=12ld=24 t=1.3779 P=0.0344

40

3.5 Notch

In situ for the candidate gene Notch was performed with the purpose to

check neural stem cells, since GFAP is only a marker for glial

characteristics.

The result showed expression of Notch only in the cells boarding the third

ventricle as expected, but there was also a relevant increase of signal in the

injured tissue, seemingly in contradiction with the known role of Notch.

Morover, in the injured samples tested by sense probe was found

widespread signal. An eventual unspecific binding of the probe is expected

to present the same spread distribution in all similar samples, while Notch

presented it only for the sense probe in the injured material.

It was supposed that Notch transcription involves also the complementary

strand with the production of an antisense mRNA. These possibility was

however not evaluated further because of time constrictions.

Figure 16In situ hybridization of Notch antisense probe in adult salamander midbrain

41

4 Discussion

On three antisense probes for the candidate genes, two of them worked and

permitted to analyze the mRNA expression of the related genes. Both the

probes, nRAD and Jar2, permitted to show a differential expression

between the control and the injured samples.

Some consideration about the result obtained by stem marker Notch

compared with its role are needed.

4.1 Notch

Notch is known to decrease neuronal differentiation and proliferation

(Chambers 2001) and so to maintain stemness, so it would not be expected

to discover up-regulation of Notch in a regenerating tissue where

proliferation and neuronal differentiation are in act. Instead the result of in

situ hybridization seems to contradict this role. Further experiments are

required to confirm the up-regulation of Notch, expecially a quantitative

analysis such as qRT-PCR.

However the regulation of Notch in the injured tissue appears to be

different zone by zone, in fact there is not expression of Notch in the cells

far from the third ventricle where neuronal differentiation is in act, but the

42

43

signal increases in the cells edging the ventricle that are known to have

stem and glial properties.

Since the ependymoglial cells are stem cells, it is hypothesized that Notch

might play a role in gliogenesis, promoting proliferation of the

ependymoglia during tissue repair. PCNA expression in tissues 7 days after

6-OHDA injection could be a confirmation of this hypothesis, since a

relevant number of PCNA positive cells were from the ependymoglial layer.

4.2 nRAD

The evident nRAD expression in tissue 7 days after lesion, indicates the

involvement of this gene in adult brain regeneration, as muscle

regeneration, so to let think a parallelism between regenerative pattern of

the two structures can really exist.

The role of nRAD in regeneration (muscular or neuronal) is not yet known,

so it is not possible to speculate about experimental results compared with

its role. Anyway the exact localization of nRAD positive cells in injured

tissue so close with PCNA positive cells, suggests this gene could be

involved in proliferation of new cells. Also the little size of PCNA positive

cells gathered in the expression area of nRAD let think about a massive

proliferation of new cells directly linked with nRAD gene, supporting this

hypothesis.

However, to test it needs dedicated studies.

44

4.3 JARID2

Decreasing of Jar2 expression in injured tissues revealed by in situ

hybridization, indicates the involvement of JARID2 in the regenerative

pattern of adult vertebrate brain.

Quantitative analysis of JARID2 protein expression induces to confirm the

same result as in situ: there is a significative down-regulation in injured

tissues.

Moreover a qRT-PCR analysis performed in the same laboratory by a

parallel research individuated the same down-regulation of JARID2 in

6-OHDA treated tissues (personal communications).

A decrease of JARID2 expression in regenerating tissues is probably

necessary in order to avoid silencing of genes involved in proliferation

and/or differentiation by Polycomb repressive group 2, and to allow in this

way the correct processes for tissue repair.

The epigenetic regulation pattern could present differences between

uninjured and regenerating tissues, and show eventual targets of

transcription activated by epigenetic modifications in 6-OHDA injured

brains.

It would be required a Chromatine Immunoprecipitation in order to study

the epigenetic methylation pattern and check if some genes, known to be

up-regulated or activated 7 days after 6-OHDA injection, are influenced.

However this experimentation is subordinated to the study about genes

identified by micro array transcription assay.

Instead, focusing the attention on genes transcriptional levels at different

stages of the regenerating process, would allow to individuate which genes

regulate expression and activity of JARID2.

Then, after would be possible to individuate the genes involved in

recognizing neuronal injuries and consequent activation of regenerating

processes leading to complete tissue repair.

4.4 Conclusion

The obtained results and the perspectives born from them, represent the

work path open toward the possibility to promote neural regeneration in

adult brain of vertebrates and to understand why physiologically it does not

happen in mammals.

45

46

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53

I wish to say thanks

To Andras Simon who accepted me in his lab in Stockholm giving me the opportunity to know scientific

research from a different point of view;

To Matthew Kirkham, my supervisor, who took care of me and put up with all my several questions even while he

was concentrate in working on different problems;

To Heng Wang who received me when I arrived in Stockholm and found an accomodation for my first

months in Sweden so that I didn't freeze in the winter;

To Daniel Berg, Paula Borg and Sara Loof who were always present in the office to solve any doubts or just to

take a break with conversation and swedish fika;

To Prof.ssa Renata Batistoni who helped me in relating to international research community and encouraged me

continuously;

To my father and my mother who granted me economical support to survive during all period of my education, and

above all taught me to be independent, a very precious lesson useful to live alone in a foreign country;

To Alvaro, Rob, Nancy, Johanna, Olga, Anders and all other friends known in Sweden who made my stay period

more funny, comfortable and coloured;

To Pisa, all professors, associates and students who were part of my education or just of the concurrent life time,

also the ones who never came to visit me in Sweden;

To all my family and my friends that concurred in my realisation as now I am and I work.

Marco Natali

Marco Natali Master Thesis

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